Methods of use of a ductal carcinoma antigen

ABSTRACT

Monoclonal antibodies to adenocarcinoma cells, and, in particular, breast carcinoma cells, are produced by a hybridoma formed by fusing mouse lymphocytes and mouse myeloma cells. The monoclonal antibodies are capable of shrinking solid tumors associated with human breast. The monoclonal antibodies identify an antigen associated with carcinomas of ductal lineage. The monoclonal antibodies, specifically, F36/22 monoclonal antibodies, can be used diagnostically and therapeutically.

CROSS-REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 08/733,631, filed Oct. 17,1996, currently allowed, which in turn is a divisional of applicationSer. No. 07/408,817, filed Sept. 18, 1989, now U.S. Pat. No. 5,62,114,which in turn is a divisional of application of Ser. No. 06/775,062,filed Sep. 11, 1985, currently U.S. Pat. No. 4,939,240, which in turn isa continuation-in-part of application Ser. No. 06/472,222, filed Mar. 4,1983, presently abandoned.

TABLE OF CONTENTS

1. Introduction

2. Background of the Invention

2.1. Monoclonal Antibodies

2.2. Application of Monoclonal Antibodies

2.3 Monoclonal Antibodies to Mammary Cells

3. Summary of the Invention

4. Brief Description of the Figures

5. Description of the Invention

5.1. The Antigen

5.2. Somatic Cells

5.3. Myeloma Cells

5.4. Fusion

5.5. Isolation of Clones and Antibody Detection

5.6. Cell Propagation and Antibody Production

5.7. In Vitro Diagnostic Uses for Monoclonal Antibodies to Human BreastCarcinoma

5.7.1. Immunohistological and Immunocytological Applications

5.7.2. Immunoserological Applications

5.8. In Vivo Diagnostic and Therapeutic Uses for Monoclonal Antibodiesto Human Breast Carcinoma

5.8.1. Tumor Localization

5.8.2. Passive Immunotherapy for Treatment of Human Cancer

5.8.3. Treatment of Human Cancer with Monoclonal Antibody Conjugates

5.9. Active Immunotherapy for Treatment of Human Cancer

6. Examples

6.1. Breast Cell Lines and Tissues

6.2. Immunization and Cell Fusion

6.3. Isotyping of F36/22 Monoclonal Antibodies

6.4. Immunoglobulin Preparations

6.5. Cell-Surface Radioimmunoassay (CS-RIA)

6.6. In Vitro Characterization of Monoclonal Antibody F36/22

6.6.1. Detection and Binding of McAb F36/26 to Cultured Cells

6.6.2. Quantitative Adsorption of McAb F36/22 to Cultured Cells

6.6.3. Reciprocal Binding Inhibition

6.6.4. In Vitro Cytotoxicity of McAb F36/22

6.6.5. Reactivity of McAb F36/22 with Estrophilin Complexes

6.6.6. Relationship Between Estradiol Concentration and McAb F36/22Binding Levels

6.7. Characterization of the Antigen Recognized by Monoclonal AntibodyF36/22

6.7.1. Solid-Phase Adsorption of BT-20 Breast Carcinoma Antigen byLectins

6.7.2. Antigen Modulation

6.7.3. Release of Antigen Recognized by McAb F36/22 by Tumor Cells

6.7.4. Gel Filtration Chromatography of MCF-7 Antigen Recognized by McAbF36/22

6.7.5. Effect of Enzyme Digestions on Cell Surface Antigenicity

6.8. Detection of Ductal Carcinoma Antigen in Breast Cancer Sera UsingMonoclonal Antibody F36/22

6.8.1. Enzyme Immunoassay Procedure

6.8.2. Biochemical Characteristics of Circulating Sera

6.8.3. Solid Phase Adsorption of Serum Antigen

6.9. Immunoaffinity Isolation of Ductal Carcinoma Antigen UsingMonoclonal Antibody F36/22

6.9.1. Purification of Antigen

6.9.2. Size Fractionation of Antigen

6.9.3. Polyacrylamide Gel Electrophoresis

6.9.4. Enzyme Immunoassay

6.9.5. Physical Characteristics of Purified Antigen

6.9.6. Lectin Binding Ability of Purified Antigen

6.9.7. Effects of Physical Treatment, Chemical Modifications and Enzymeson the Binding of McAb F36/22 to Purified Antigen

6.10. In Vitro Immunohistological Applications of Monoclonal AntibodyF36/22

6.10.1. Immunoperoxidase Staining of Tumor Specimens by McAb F36/22

6.10.2. Immunoreactivity of McAB F36/22 With Normal Mammary Tissue,Membrane Preparations and Milk

6.10.3 Immunoperoxidase Staining of Human Breast Tissues and Tumors

6.10.4. Effect of Varying McAb F36/22 Concentration and Incubation Timeson Immunoperoxidase Staining Results

6.10.5. Estrogen Receptor Levels and Immunoperoxidase Staining

6.11. In Vivo Applications of Monoclonal Antibody F36/22

6.11.1. Experimental Induction of Solid Tumors in Mice

6.11.2. McAb F36/22 Targeting: In Vivo Tumor Localization

6.11.3. In Vivo Passive Immunotherapy with McAb F36/22

1. INTRODUCTION

This invention relates to the production of and applications formonoclonal antibodies specific for tumor antigens. More particularly,this invention relates to monoclonal antibodies against cell surfaceantigenic determinants expressed maximally on breast carcinomas and alsopresent on other adenocarcinomas. Monoclonal antibodies capable ofreacting with cell-surface antigens are of value for theimmunoclassification and detection of disease and represent novel agentsfor immunotherapy. The monoclonal antibodies of this invention possessdistinctive characteristics and capabilities which make them suitablefor in vitro clinical diagnostic purposes. Moreover, they are of greatpotential importance for in vivo tumor localization and cancer therapyin humans.

The monoclonal antibodies exhibit a high level of binding to breastcarcinoma cells and are capable of experimental in vivo tumorlocalization. They bind to well-differentiated as well as topoorly-differentiated tumors. The antigen recognized is a cell surfacecomponent associated with carcinomas of ductal lineage that remainsunmodulated after exposure to antibody. The monoclonal antibodiesexhibit the ability to fix human complement. Most importantly, themonoclonal antibodies of this invention are capable of inducing a rapidand significant volume reduction of established, progressively growing,solid human breast tumors xenografted in animals (mice). The tumoricidaleffectiveness is quite remarkable with tumor volume shrinkage of greaterthan 70% in short intervals (three days) after administration of lowdoses (100 μg) of antibody. Immunotherapy occurs passively,independently of any cytotoxic compounds.

The invention provides methods for production of the monoclonalantibodies by hybridoma techniques. Once cloned, cell lines can bemaintained continuously to produce an unlimited homogeneous monoclonalantibody population that can be isolated and/or purified and usedclinically for in vitro immunohistological, immunocytological orimmunoserological diagnosis, in vivo diagnosis by localization of tumorsand metastases, and immunotherapy of human cancers, particularly thoseof the breast.

2. BACKGROUND OF THE INVENTION 2.1. Monoclonal Antibodies

Kohler and Milstein are generally credited with having devised thetechniques that successfully resulted in the formation of the firstmonoclonal antibody-producing hybridomas G. Kohler and C. Milstein,Nature 256:495-497 (1975); Eur. J. Immunol. 6:511-519 (1976)!. By fusingantibody-forming cells (spleen lymphocytes) with myeloma cells(malignant cells of bone marrow primary tumors) they created a hybridcell line, arising from a single fused cell hybrid (called a hybridomaor clone) which had inherited certain characteristics of both thelymphocytes and myeloma cell lines. Like the lymphocytes (taken fromanimals primed with sheep red blood cells as antigen), the hybridomassecreted a single type of immunoglobulin specific to the antigen;moreover, like the myeloma cells, the hybrid cells had the potential forindefinite cell division. The combination of these two features offereddistinct advantages over conventional antisera. Whereas antisera derivedfrom vaccinated animals are variable mixtures of polyclonal antibodieswhich never can be reproduced identically, monoclonal antibodies arehighly specific immunoglobulins of a single type. The single type ofimmunoglobulin secreted by a hybridoma is specific to one and only oneantigenic determinant, or epitope, on the antigen, a complex moleculehaving a multiplicity of antigenic determinants. For instance, if theantigen is a protein, an antigenic determinant may be one of the manypeptide sequences generally 6-7 amino acids in length (M. Z. Atassi,Molec. Cell. Biochem. 32:21-43 (1980)! within the entire proteinmolecule. Hence, monoclonal antibodies raised against a single antigenmay be distinct from each other depending on the determinant thatinduced their formation; but for any given clone, all of the antibodiesit produces are identical. Furthermore, the hybridoma cell line can bereproduced indefinitely, is easily propagated in vitro or in vivo, andyields monoclonal antibodies in extremely high concentration.

2.2. Application of Monoclonal Antibodies to Cancer

Monoclonal antibodies presently are being applied by investigators tothe diagnosis and treatment of cancer For a general discussion of thetopic, see Hybridomas in Cancer Diagnosis and Treatment, Mitchell, M. S.and Oettgen, H. F., (eds.), Progress in Cancer Research and Therapy,Vol. 21, Raven Press, New York (1982)!. It has been reported thatmonoclonal antibodies have been raised against tumor cells U.S. Pat. No.4,196,265!, carcinoembryonic antigen U.S. Pat. No. 4,349,528!, andthymocytes, prothymocytes, monocytes and suppressor T cells U.S. Pat.Nos. 4,364,933; 4,364,935; 4,364,934; 4,364,936; 4,364,937; and4,364,9321!. Recent reports have demonstrated the production ofmonoclonal antibodies with various degrees of specificity to severalhuman malignancies, including mammary tumor cells Colcher, D. et al.,Proc. Natl. Acad. Sci. U.S.A. 78:3199-3203 (1981)!, lung cancersCuttitta, F. et al., Proc. Natl. Acad. Sci: U.S.A. 78:495-4595 (1981)!malignant melanoma Dippold, W. G. et al., Proc. Natl. Acad. Sci. U.S.A.,77:6114-6118 (1980)!, 1, colorectal carcinoma Herlyn, M. et al., Proc.Natl. Acad. Sci. U.S.A. 76:1438-1442 (1979)), lymphoma Nadler, L. M. etal., J. Immunol. 125:570-577 (1980)!, and neuroectodermal tumorsWikstrand, C. J. and Bigner, D. C., Cancer Res., 42:267-275 (1982)!.

Investigators have noted the potential immunotherapeutic value ofmonoclonal antibodies and some investigators have investigatedtherapeutic efficacy in both animal and human subjects Miller, R. A. etal., New Eng. J. Med. 306:517-522 (1982); Ritz, J. and Schlossman, S.,Blood, 59:1-11 (1982); and Kirch, M. E. and Ulrich, H. J. Immunol.,127:805-810 (1981)!. Although most studies have described the effects ofcytotoxic drug-antibody conjugates, Beverly, P. C. L., Nature, 297:358-9(1982); Krolick, K. A. et al., Nature, 295:604-5 (1982); Krolick, K. A.et al., Proc. Natl. Acad. Sci. U.S.A., 77:5419-23 (1980); Arnon, R. andSela, M.; Immunol. Rev., 62:5-27 (1982); Raso, V. et al., Cancer Res.,42:457-64 (1982); and DeWeger, R. A. and Dullens, H. F. J., Immunol.Rev. 62:29-45 (1982)!, experimental and clinical studies on passiveimmunotherapies have been attempted Sears, H. F. et al., Lancet,i:762-65 (1982); Rosenberg, S. A. and Terry, W. D., Cancer Res.,25:323-88 (1977); Herlyn, D. M. et al., Cancer Res., 40:717-21 (1980);Scheinberg, D. A. and Strand, M., Cancer Res. 42:44-9 (1982); Koprowski,H. et al., Proc. Natl. Acad. Sci. U.S.A., 75:3405-9 (1978); and Young,Jr., W. W. and Hakomori, S.-I., Science, 211:487-9 (1981)!. In theexperimental setting, however, most studies have dealt with theconcurrent administration of monoclonal antibody and tumor inoculum, oradministration within several days of the implantation of tumor cells,resulting in a decreased tumor take or growth rate of xenografts. Inthis context, these data form a basis for the immunoprophylaxis of tumordevelopment. It is an object of the present invention to providemonoclonal antibodies demonstrating a therapeutic effect onprogressively-growing established tumors. To our knowledge, prior tothis invention, tumor volume reduction against well establishedprogressively growing solid tumors with the use of passive monoclonalantibody has not been reported.

2.3 Monoclonal Antibodies to Mammary Cells

Several investigators have reported on the production of monoclonalantibodies against epitopes of various normal and malignant mammary cellcomponents. Arklie, J. et al., Int. J. Cancer, 28:23-29(1981); Ciocca,D. R. et al., Cancer Res., 42:4256-4258(1982); Colcher, D. et al., Proc.Natl. Acad. Sci., U.S.A. 78:3199-3203 (1981); Foster, C. S. et al.,Virchows Arch. Pathol. Anat., 394:279-293(1982); Greene, G. L. et al.,Proc. Natl. Acad. Sci. U.S.A., 77:5115-5119(1980)!; McGee, JO'D. et al.,Lancet 2:7-11(1982); Nuti, M. et al., Int. J. Cancer, 291:539-545(1982);and Taylor-Papadimitriou, J. et al., Int. J. Cancer, 28:17-21(1981)!.Many of the antigens recognized above are differentiation-related;therefore these antibodies are most suited to histologically assess thedifferentiated status or grade of tumor specimens. For example,monoclonal antibodies directed against several antigens of humanmilk-fat-globule membranes have been produced. These antibodies haveproven useful in studying the derivation of cell cultures, in evaluatingthe phenotypic expression of antigens in neoplastic transformation, andhave served as differentiation markers in breast cancer, and asimmunodiagnostic reagents in the quantitation of antigens in the sera ofbreast cancer patients Arklie, J. et al., Int. J. Cancer,28:23-29(1981); Ceriani, R. L. et al., Proc. Natl. Acad. Sci. U.S.A.,74:582-586(1977); Ceriani, R.L. et al., Proc. Natl. Acad. Sci.,79:5420-5424(1982); Foster, C. S. et al., Virchows Arch. Pathol. Anat.,394:279-293(1982); and Taylor-Papadimitriou, J. et al., Int J. Cancer,28:17-21(1981)!. However, there is further need for monoclonalantibodies which are differentiation-unrelated and of use to detectantigen occurring in poorly-differentiated breast tumors. It is anobject of this invention to provide such monoclonal antibodies. Suchreagents may reflect subtle biochemical or antigenic differences inbreast cancer, independent of tumor grade. The use of such antibodiescan add significant information regarding functional classifications ofindividual breast tumors to augment clinical classifications.

The pattern of staining for the monoclonal antibody of this invention isdistinct from the reactivities of previously described monoclonalantibodies which recognize antigens expressed by breast tumors. Arklieet al. supra! have described monoclonal antibodies directed againsthuman milk-fat-globule membranes. These antibodies showed a strongerstaining reaction with well-differentiated (grade I) ductal carcinomasthan undifferentiated (grade III) tumors. By comparison, the monoclonalantibody provided herein stained poorly-differentiated tumors equallywell as well-differentiated breast carcinoma specimens. Therefore, themonoclonal antibody identifies a sub-class of breast carcinomas which ishistologically indistinguishable from tumors lacking antigen expression.Nuti et al. supra! have produced monoclonal antibodies against humanmetastatic breast carcinoma cells which have been used to indicate tumorantigen heterogeneity, but which failed to react with normal and benignmammary tissues or uterus. Other reported monoclonal antibodies directedagainst carcinoma-associated antigens McGee et al., supra! also did notreact with any benign conditions of the breast and very few normaltissues. Foster et al. supra! have also reported the production ofmonoclonal antibodies which were used to show significant heterogeneityof antigen expression within breast tumors, but whose specifities aredistinct from the monoclonal antibodies of the present invention.

3. SUMMARY OF THE INVENTION

Prior to the present invention, applicants believe there has been noreport of a clinically useful preparation of monoclonal antibodieswhich, upon passive administration, is capable of reducing the volume ofwell established, progressively growing solid tumors associated with thehuman breast. In fact, it is applicants' belief that volume reduction ofany other established and growing solid tumors with the use of passivemonoclonal antibody immunotherapy has not been reported. The presentinvention provides methods and compositions for producing novelmonoclonal anti-breast carcinoma antibodies with specific binding,cytotoxic and tumor shrinkage capabilities and encompasses the use ofsaid antibodies for cancer immunodiagnosis and passive immunotherapy inhumans.

Specifically, the invention provides novel hybridoma-derived monoclonalantibodies which react with a determinant (epitope) located both ondifferentiated mammary duct epithelia and on certain breast carcinomas;the monoclonal antibodies do not react with estrophilin, bone marrowstem cells or lymphoid cells. In contrast to the expression ofpreviously described differentiation antigens, the epitope recognized bythe monoclonal antibodies of this invention is also found in a highpercentage of poorly-differentiated carcinomas of the human breast. Theextent of antigenic expression, as detected by the monoclonalantibodies, is similar for well and poorly differentiated tumors and isindependent of the histological characteristics of the tumor. Themonoclonal antibodies can be used immunologically to sub-classify tumorswhich are histologically indistinguishable by conventionalhistopathological staining techniques and to establish phenotyping ofbreast cancer. Hence, the monoclonal antibodies of the present inventionrepresent new in vitro immunodiagnostic reagents for the early andaccurate detection of certain cancers.

The epitope recognized by the monoclonal antibody of this invention isalso present on other adenocarcinomas, including those of the colon,ovary, uterus, pancreas and prostate (see Section 6.10.1), although theantigenic determinant is expressed at a lower level than in breastcancer. The normal tissue of these histotypes (colon, ovary, uterus,pancreas, prostate, etc.) contain no detectable levels of thedeterminant. Therefore, the expression of the antigen recognized by themonoclonal antibodies may be associated with the neoplastic developmentof these histotypes. Hence, the monoclonal antibodies of the presentinvention represent a new-diagnostic indicator of certain cancers inthese tissues. Because certain adenocarcinomas do not contain detectablelevels of the epitope, the monoclonal antibodies identify antigenicdifferences which can be prognostically significant.

In addition to their use as in vitro immunohistological reagents forcancer diagnosis, the monoclonal antibodies of the present invention canbe used diagnostically in vivo. Because of their ability to targetbreast carcinoma cells in vivo, the monoclonal antibodies can be used intumor localization and in the monitoring of metastases.

Furthermore, the monoclonal antibodies provided herein can be used as invitro immunoserological and immunocytological reagents on body fluids todetect the presence of the specific antigen and/or cells bearingantigen. The monoclonal antibodies thereby permit non-invasive diagnosisof certain breast carcinomas and other cancers.

The present invention also contemplates the use of the monoclonalantibodies for serodetection of ductal carcinoma antigen to be used inthe differential diagnosis of certain carcinomas of ductal lineage.

Most importantly, the hybridoma-derived monoclonal antibodies of thepresent invention represent new immunotherapeutic agents for thetreatment of human breast cancer. The monoclonal antibodies are capableof inducing a rapid and significant reduction of progressively growingadenocarcinomas associated with the human breast. The tumoricidaleffectiveness at low doses of the monoclonal antibodies, the highincidence of antigen expression in biopsies of breast cancer patientsand the high specific binding of the monoclonal antibodies to certainbreast tumors, the absence of modulation of the antigen recognized andthe ability of the monoclonal antibodies to fix human complement anderadicate cancer cells independent of other cytotoxic agents areindications of the usefulness of the monoclonal antibodies in humanbreast cancer therapy.

The present invention provides methodologies useful in research for theevaluation of parameters associated with the use of monoclonalantibodies against human tumors in passive human immunotherapy. Themonoclonal antibodies can be used as probes to investigate the roles ofantigen density, tumor growth rates, tumor size, cellular heterogeneityand other variables in the susceptibility of tumors to immunotherapy.

The invention contemplates the use of the monoclonal antibodies providedherein in covalent combination with cytotoxic or chemotherapeuticmolecules. For instance, the monoclonal antibodies can be conjugated tocertain cytotoxic compounds, including, but not limited to, radioactivecompounds, diphtheria toxin (chain A), ricin toxin (chain A),adriamycin, chlorambucil or daunorubicin, to enchance their tumoricidaleffectiveness. The invention also contemplates the covalent combinationof the monoclonal antibodies with carbohydrate-active reagents such as,but not limited to, glycosidases, to unmask the cell surface antigenrecognized, thereby increasing antibody binding and tumoricidaleffectiveness.

The present invention further contemplates the use of the monoclonalantibodies in immunoadsorption procedures to effectively separate breastcancer cells from marrow elements based upon antibody binding, and incomplement-mediated cytolytic procedures to eliminate malignant cellswhile sparing bone marrow stem cells. Because such procedures require atumor antigen, such as the one recognized by the monoclonal antibodiesof this invention, which is not found on bone marrow stem cells orlymphoid cells, the monoclonal antibodies represent a reagent useful foreliminating disseminated breast cancer cells from autologous bonemarrow.

Because the monoclonal antibodies are produced by hybridoma techniques,the present invention provides theoretically immortal cell lines capableof consistently producing high titers of single specific antibodiesagainst a distinct breast carcinoma antigen. This is a distinctadvantage over the traditional technique of raising antibodies inimmunized animals where the resulting sera contain multiple antibodiesof different specificities that vary in both type and titer with eachanimal, and, in individual animals, with each immunization.

The invention further provides purified ductal carcinoma antigen, all orpart of which antigen may serve as the basis for a vaccine againstcancers characterized by the presence of tumor cells expressing theantigen. The invention also contemplates active immununotherapiesinvolving the administration to humans of such a vaccine. The inventionfurther comtemplates preparing anti-idiotype antibodies to themonoclonal antibodies of this invention and preparing anti-idiotypevaccines to be administered to humans for treatment of cancer.

The invention further provides purified ductal carcinoma antigen, all orpart of which antigen may serve as the basis for a vaccine againstcancers characterized by the presence of tumor cells expressing theantigen. The invention also contemplates active immunotherapiesinvolving the administration to humans of such a vaccine. The inventionfurther contemplates preparing anti-idiotype antibodies to themonoclonal antibodies of this invention and preparing anti-idiotypevaccines to be administered to humans for treatment of cancer.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1, graphically illustrates solid-phase adsorption of serum antigen.Specimens of breast cancer sera (N=6) were allowed to react with varioussolid-phase adsorbents. Nonderivatized Sepharose served as a negativecontrol (i.e., 0% bound antigen). After incubation, each supernatant wasassayed for antigen activity (triplicate measurements), and this valuewas used to calculate the percentage of bound antigen. McAbF36/22:Sepharose was used as a positive control for these experiments.Binding specificities: concanavalin A, α-D-mannosyl groups; wheat germlectin, β-N-acetylglucosaminyl groups; peanut agglutinin,β-N-acetylglucosaminyl groups; Protein A, Fc₆₅ -immunoglobulin domain.McAb F5 reacts with an antigen specific for prostate tissue. Statisticalevaluations (Student's t test) indicated that the solid-phase adsorbentsprepared from wheat germ lectin and McAb F36/22 bound statisticallysignificant (p less than 0.01) amounts of serum antigen as compared tocontrol Sepharose.

FIG. 2, graphically illustrates molecular sieve chromatography ofmalignant effusions obtained from patients with breast cancer (BCA) orovarian cancer (OCA). Fractions were evaluated for protein (absorbanceat 280 nm) and antigen content (absorbance at 480 nm). In all fluidsstudied peak antigen activity eluted in the included volume, ahead ofthe 669,000 molecular weight marker protein. The column wasprecalibrated using blue dextran void volume (V_(o))!, bovinethyroglobulin (669 KD), bovine catalase (240 KD) and phenol red totalbed volume (V_(t))!.

FIG. 3. graphically illustrates an enzyme immunoassay of theimmunoaffinity- purified ductal carcinoma antigen from malignanteffusion. The assay was performed in the presence of standard assaybuffer containing pH 7.0 phosphate salts or borate salts at pH 7.0.

FIG. 4. graphically illustrates the stability of immunopurified antigenafter acid, base and heat treatment. Antigen was incubated for 5 hoursat 60° C. in the presence or absence of 0.1N NaOH or 0.1N HCl. Residualactivity was determined using an enzyme immunoassay procedure(absorbance at 488 nm).

5. DESCRIPTION OF THE INVENTION 5.1. The Antigen

In the embodiment of the invention described in Section 6.2., MCF-7breast carcinoma cells were used as `antigen`. However, as demonstratedby experiments described in Section 6.6.1., 6.10.1., and 6.10.3., theepitope recognized by the antibody of this invention is present on thesurface of several cultured cell lines as well as cells present incertain extra-mammary tumors. Hence, these cells also representpotential `antigen` or sources of antigen with which to immunize animalsto obtain somatic cells for fusion.

For example, the BT-20, MDA-MB-157, and to a lesser extent, SK-BR-3breast carcinoma cell lines can be used as immunogens. Non-breastcarcinoma cell lines, including PANC-1 (pancreas carcinoma) and HT-29(colon carcinoma) also represent potential candidates as immunogen inother embodiments of the invention. Furthermore, cells derived fromintensely-staining benign or malignant breast tissues listed in TABLEXI, can be used. Similarly, cells derived from positively-stainingextra-mammary tumors listed in TABLE XI, for instance ovarian, colon,endometrial, renal, bronchogenic, etc., carcinomas or ovariancystadenomas, can be used as immunogen.

Cells or fluids derived from pleural effusions of breast cancer patients(see Section 6.7.3.) and antigen isolated from cell lysates as describedin Section 6.7.4. are also potential candidates with which to immunizeanimals and humans.

In the embodiment of the invention described herein, monoclonalantibodies raised against a breast tumor line identified an antigenassociated with carcinomas of ductal lineage and occuring on a limitednumber of normal ductal elements. The antigen, hereinafter referred toas ductal carcinoma antigen (DCA), has been detected in human bodyfluids such as malignant effusions, lymph, sera and thoracentesis.Electrophoretic analysis of the antigen demonstrated the isolation of asingle high molecular weight glycoprotein exhibiting an isoionic pointnear pH 4.2 and a density of approximately 1.45/ml. Although highlyreactive with wheat germ lectin, a negligible or weak interaction wasobserved with concanvalin A, lentil lectin and peanut agglutinin. Theantigen was immune-precipitable, indicating the occurence of multiplemonoclonal antibody-binding sites, and was resistant to heat and acidtreatments. Antigenicity was not perturbed following protease orneuraminidase treatments, but was affected upon exposure to alkalineconditions. These data suggest that monoclonal antibody F36/22recognizes a high molecular weight component occurring in circulation asa mucin-like glycoprotein (see Section 6.8).

5.2. Somatic Cells

Somatic cells with the potential for producing antibody and, inparticular B lymphocytes, are suitable for fusion with a B-cell myelomaline. Those antibody-producing cells that are in the dividingplasmablast stage fuse preferentially. Somatic cells may be derived fromthe lymph nodes, spleens and peripheral blood of primed animals and thelymphatic cells of choice depends to a large extent on their empiricalusefulness in the particular fusion system. Once- primed orhyperimmunized animals can be used as a source of antibody-producinglymphocytes. Mouse lymphocytes give a higher percentage of stablefusions with the mouse myeloma lines described in Section 5.3. However,the use of rat, rabbit, and frog cells is also possible.

Alternatively, human somatic cells capable of producing antibody,specifically B lymphocytes, are suitable for fusion with myeloma celllines. While B lymphocytes from biopsied spleens or lymph nodes ofindividuals may be used, the more easily accessible peripheral blood Blymphocytes are preferred. The lymphocytes may be derived from patientswith diagnosed breast or other adenocarcinomas.

5.3. Myeloma Cells

Specialized myeloma cell lines have been developed from lymphocytetumors for use in hybridoma-producing fusion procedures G. Kohler and C.Milstein, Eur. J. Immunol. 6:511-519 (1976); M. Schulman et al., Nature276:269-270 (1978)!. The cell lines have been developed for at leastthree reasons. The first is to facilitate the selection of fusedhybridomas among unfused and similarly indefinitely self-propogatingmyeloma cells. Usually, this is accomplished by using myelomas withenzyme deficiencies that render them incapable of growing in certainselective media that support the growth of hybridomas. The second reasonarises from the inherent ability of lymphocyte tumor cells to producetheir own antibodies. The purpose of using monoclonal techniques is toobtain immortal fused hybrid cell lines that produce the desired singlespecific antibody genetically directed by the somatic cell component ofthe hybridoma. To eliminate the production of tumor cell antibodies bythe hybridomas, myeloma cell lines incapable of producing light or heavyimmunoglobulin chains or those deficient in antibody secretionmechanisms are used. A third reason for selection of these cell lines isfor their suitability and efficiency for fusion.

Several myeloma cell lines may be used for the production of fused cellhybrids, including X63-Ag8, NSI-Ag4/1, MPC11-45.6TG1.7, X63-Ag8.653,Sp2/0-Ag14, FO, and S194/5XX0.BU.1, all derived from mice,210.RCY3.Ag1.2.3 derived from rats and U-226AR, and GM1500GTGAL₂ derivedfrom rats and U-226AR, and GM1500GTGAL₂, derived from humans. G. J.Hammerling, U. Hammerling and J. F. Kearney (eds.), Monoclonalantibodies and T-cell hybridomas In J. L. Turk (ed.) Research Monographsin Immunology, Vol. 3, Elsevier/North Holland Biomedical Press, New York(1981)!.

5.4Fusion

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion as in the example in Section 6.2.(though the proportion may vary from about 20:1 to about 1.:l),respectively, in the presence of an agent or agents (chemical orelectrical) that promote the fusion of cell membranes. It is oftenpreferred that the same species of animal serve as the source of thesomatic and myeloma cells used in the fusion procedure. Fusion methodshave been described by Kohler and Milstein Nature 256:495-497 (1975) andEur. J. Immunol. 6:511-519 (1976)!, and by Gefter et al. Somatic CellGenet. 3:231-236 (1977)). The fusion-promoting agent used by thoseinvestigators were Sendai virus and polyethylene glycol (PEG),respectively. The fusion procedure of the example of the presentinvention is a modification of the method of Kohler and Milstein, supra.

5.5. Isolation of Clones and Antibody Detections

Fusion procedures usually produce viable hybrids at very low frequency,about 1×10⁻⁶ to 1×10⁻⁸. Because of the low frequency of obtaining viablehybrids, it is essential to have a means to select fused cell hybridsfrom the remaining unfused cells, particulary the unfused myeloma cells.A means of detecting the desired antibody-producing hybridomas among theother resulting fused cell hybrids is also necessary.

Generally, the fused cells are cultured in selective media, for instanceHAT medium containing hypoxanthine, aminopterin and thymidine. HATmedium permits the proliferation of hybrid cells and prevents growth ofunfused myeloma cells which normally would continue to divideindefinitely. Aminopterin blocks de novo purine and pyrimidine synthesisby inhibiting the production of tetrahydrofolate. The addition ofthymidine bypasses the block in pyrimidine synthesis, while hypoxanthineis included in the media so that inhibited cells can synthesize purineusing the nucleotide salvage pathway. The myeloma cells employed aremutants lacking hypoxanthine phosphoribosyl transferase (HPRT) and thuscannot utilize the salvage pathway. In the surviving hybrid, the Blymphocyte supplies genetic information for production of this enzyme.Since B lymphocytes themselves have a limited life span in culture(approximately two weeks), the only cells which can proliferate in HATmadia are hybrids formed from myeloma and spleen cells.

To facilitate screening of antibody secreted by the hybrids and toprevent individual hybrids from overgrowing others, the mixture of fusedmyeloma and B lymphocytes is diluted in HAT medium and cultured inmultiple wells of microtiter plates. In two to three weeks, when hybridclones become visible microscopically, the supernatant fluid of theindividual wells containing hybrid clones is assayed for specificantibody. The assay must be sensitive, simple and rapid. Assaytechniques include radioimmunoassays, enzyme immunoassays, cytotoxicityassay, and plaque assays.

5.6. Cell Propagation and Antibody Production

Once the desired fused cell hybrids have been selected and cloned intoindividual antibody-producing cell lines, each cell line may bepropagated in either of two standard ways. A sample of the hybridoma canbe injected into a histocompatible animal of the type that was used toprovide the somatic and myeloma cells for the original fusion. Theinjected animal develops tumors secreting the specific monoclonalantibody produced by the fused cell hybrid. The body fluids of theanimal, such as serum or ascites fluid, can be tapped to providemonoclonal antibodies in high concentration. Alternatively, theindividual cell lines may be propogated in vitro in laboratory culturevessels; the culture medium, also containing high concentrations of asingle specific monoclonal antibody, can be harvested by decantation,filtration or centrifugation.

5.7. In Vitro Diagnostic uses for Monoclonal Antibodies to Human BreastCarcinoma 5.7.1. Immunohistological and Immunocytological Applications

The monoclonal antibodies of this invention can be used as probes indetecting discrete antigens in human tumors. The expression or lack ofexpression of these antigens can provide clinically exploitableinformation which is not apparent after standard histopathologicalevaluations. It may thus be possible to correlate the immuno-phenotypesof individual tumors with various aspects of tumor behavior andresponsiveness to certain types of therapies, thus establishingimportant classifications of prognosis.

Monoclonal antibodies produced by the hybridoma methodologies hereindescribed can be used to detect potential breast carcinoma cells inhistological and cytological specimens and in particular, to distinguishwell-differentiated from poorly-differentiated tumors based on stainingpatterns and intensities. For instance, using the immunoperoxidasestaining technique described in Section 6.8.1., it has been observedthat the monoclonal antibodies of its invention stained an apicalmembrane-associated moiety in sections of benign lesions andwell-differentiated adenocarcinomas of the breast; less-differentiatedtumors however, generally exhibited a cytoplasmic staining pattern.While normal breast tissue also exhibited the surface staining patternof benign and well-differentiated lesions, the intensity of staining wasconsiderably less (See TABLE XI, Section 6.10.3.).

Another important in vitro diagnostic application of the monoclonalantibodies of this invention is the evaluation of adenocarcinomas otherthan breast. The antibody has been used to detect the epitope inadenocarcinomas of several histotypes including colon, ovary, uterus,pancreas and prostate, while the normal counterparts at thesehistological sites did not express the determinant. Thus, the novelmonoclonal antibody detects an antigenic determinant in theseadenocarcinomas which is tumor-associated. For example, no normalovaries have displayed detectable levels of the epitope, whereas 15/15ovarian carcinomas have expressed the determinant. Since the expressionof this antigenic determinant appears to be associated with neoplasticdevelopment in the ovary, the monoclonal antibody may be a usefulimmunodiagnostic reagent for ovarian carcinomas. Other adenocarcinomas(gastric, jejunal, colonic, pancreatic, prostatic and bronchogenic)exhibited varying incidences of antigen expression. Since everyadenocarcinoma specimen did not contain detectable levels of thedeterminant, the monoclonal antibody identifies antigenic differenceswhich may be prognostically significant.

As an alternative to immunoperoxidase staining, immunofluorescenttechniques can be used to examine human specimens with monoclonalantibodies to breast carcinoma. In a typical protocol, slides containingcryostat sections of frozen, unfixed tissue biopsy samples orcytological smears are air dried and incubated with the monoclonalantibody preparation in a humidified chamber at room temperature.

The slides are then layered with a preparation of antibody directedagainst the monoclonal antibody, usually some type of antimouseimmunoglobulin if the monoclonal antibodies used are derived from thefusion of a mouse spleen lymphocyte and a mouse myeloma cell line. Thisantimouse immunoglobulin is tagged with a compound that fluoresces at aparticular wavelength for instance rhodamine or fluoresceinisothiocyanate. The staining pattern and intensities within the sampleare then determined by flourescent light microscopy and optionallyphotographically recorded.

5.7.2. Immunoserological Applications

The use of the monoclonal antibodies described herein can be extended tothe screening of human biological fluids for the presence of thespecific antigenic determinant recognized. In vitro immunoserologicalevaluation of sera withdrawn from patients thereby permits non-invasivediagnosis of cancers. By way of illustration, human fluids, such aspleural fluids or lymph, can be taken from a patient and assayed for thespecific epitope, either as released antigen or membrane-bound on cellsin the sample fluid, using the anti-breast carcinoma monoclonalantibodies in standard radioimmunoassays or enzyme-linked immunoassaysknown in the art. Human sera can also be taken from a patient andassayed using standard techniques known in the art for the presence ofspecific circulating antigen recognized by the monoclonal antibodiesdescribed herein. This assay provides a simple serodiagnostic test forbreast cancer.

5.8. In Vivo Diagnostic and Therapeutic uses for Monoclonal Antibodiesto Human Breast Carcinoma 5.8.1. Tumor Localization

The monoclonal antibodies of this invention are capable of targetingbreast carcinoma cells in vivo. They can therefore be used in humans fortumor localization and for monitoring of metastases. For thisapplication, it is preferable to use purified monoclonal antibodies.Purification of monoclonal antibodies for human administration byammonium sulfate or sodium sulfate precipitation followed by dialysisagainst saline and filtration sterilization has been described by Milleret al. In Hybridomas in Cancer Diagnosis and Therapy, (1982) supra, P.134, ! and by Dillman et al. Id. at p.155! which are hereby incorporatedby reference. Alternatively, immunoaffinity chromatography techniquesmay be used to purify the monoclonal antibodies.

The purified monoclonal antibodies can be labelled with radioactivecompounds, for instance, radioactive iodine, and administered to apatient intravenously. After localization of the antibodies at the tumoror metastatic site, they can be detected by emission tomographical andradionuclear scanning techniques thereby pinpointing the location of thecancer. Experimental radioimmunodetection with monoclonal antibodies byexternal scintigraphy has been reported by Solter et al. (Id., at p.241! hereby incorporated by reference.

5.8.2. Passive Immunotherapy for treatment of Human Cancer

Because the monoclonal antibodies of this invention are capable ofinducing rapid and significant volume reduction of established,progressively growing solid tumors associated with the human breast,they may be used in the treatment of human breast cancers, both infemales and males, and possibly in the treatment of otheradenocarcinomas, particularly ovarian cancer. By way of illustration,purified anti-breast carcinoma monoclonal antibody (see Section 5.8.1)is suspended in an appropriate carrier, e.g., saline, with or withouthuman albumin, at an appropriate dosage and is administered to apatient. The monoclonal antibodies are preferably administeredintravenously, e.g., by continuous intravenous infusion over severalhours, as in Miller et al., incorporated by reference, supra. Infusionscan be administered over a period weeks during which the antitumoreffects are monitored.

The monoclonal antibodies described herein may be used clinically inconjunction with bone marrow transplantation or replacement therapies.Bone marrow tapped from patients undergoing treatment can be contactedwith the anti-breast carcinoma monoclonal antibodies to eliminatecontaminating cancer cells bearing the specific epitope byimmunoadsorption. Cells may also be eliminated by complement mediatedcytolytic procedures. Because the antigenic determinant recognized bythe anti-breast carcinoma monoclonal antibody is absent on bone marrowstem cells and lymphoid cells, the monoclonal antibodies can effectivelyremove breast carcinoma cells from autologous or allogenic bone marrowprior to transplantation or reinfusion.

5.8.3. Treatment of Human Cancer with Monoclonal Antibody Conjugates

The monoclonal antibodies of this invention can be used in conjunctionwith a broad spectrum of pharmaceutical or cytotoxic agents such as:radioactive compounds (e.g., I¹²⁵ I¹³¹ ); agents which bind DNA, forinstance, alkylating agents or various antibiotics (e.g., daunomycin,adriamycin, chlorambucil); antimetabolites such as methotrexate; agentswhich act on cell surfaces (e.g., venom phospholipases and microbialtoxins); and protein synthesis inhibitors (e.g., diphtheria toxin andtoxic plant proteins). For reviews on the subject, see Bale et al.,Cancer Research 40:2965-2972 (1980); Ghose and Blair, J. Natl. CancerInst. 61(3):657-676 (1978) Gregoriadis, Nature 265:407-411(1988);Gregoriadis, Pharmac. Ther. 10:103-108 (1980); Trouet et al., RecentResults Cancer Res. 75:229-235 (1980)!. Of particular importance arethose agents capable of exerting toxic effects at the level of the cellsurface, such as adriamycin Tritton, T. R. and Yee, G., Science,217:248-50 (1982)!.

The methods used for binding the cytotoxic agents to the monoclonalantibody molecule can involve either non-covalent or covalent linkages.Since non-covalent bonds are more likely to be broken before theantibody complex reaches the target site, covalent linkages arepreferred. For instance, carbodiimide can be used to link carboxy groupsof the pharmaceutical agent to amino groups of the antibody molecule.Bifunctional agents such as dialdehydes or imidoesters can be used tolink the amino group of a drug to amino groups of the antibody molecule.The Schiff base reaction can be used to link drugs to antibodymolecules. This method involves the periodate oxidation of a drug orcytotoxic agent that contains a glycol or hydroxy group, thus forming analdehyde which is then reacted with the antibody molecule. Attachmentoccurs via formation of a Schiff base with amino groups of the antibodymolecule. Additionally, drugs with reactive sulfhydryl groups have beencoupled to antibody molecules.

Similarly, glycosidic enzymes such as neuraminidase or α-mannosidase canbe conjugated to the monoclonal antibodies.

Conjugated antibodies can be administered to patients, as in Section5.8.2., to achieve enhanced tumoricidal effects through the cytotoxicaction of the chemotherapeutic agents or the increased binding effect ofthe glycosidase (See Section 6.7.5).

5.9. Active Immunotherapy for Treatment of Human Cancer

The physical identity of the purified antigen identified by themonoclonal antibodies of this invention also contribute to moreeffective treatment modalities based upon specific activeimmunotherapies. By way of illustration, purified ductal carcinomaantigen (DCA) is suspended in an appropriate carrier, e.g., saline, withor without human albumin at an appropriate dosage and is administered toa patient in a vaccine formulation. The antigen may act as an immunogento elicit a host immune response for the prevention and/or control oftumor growth.

The monoclonal antibodies described herein may also be used to prepareanti-idiotype antibodies that react with the antigen recognized by themonoclonal antibody. The anti-idiotype antibodies can be used in avaccine formulation for therapeutic treatment of breast cancer and otheradenocarcinomas.

6. EXAMPLES 6.1. Breast Cell Lines and Tissues

Breast carcinoma cell lines used included the following: MCF-7, derivedfrom pleural effusion of scirrhous carcinoma Soule, D. H. et al., J.Natl. Cancer Inst., 51:1409-1416 (1973) ; SK-BR-3, derived from pleuraleffusion of adenocarcinoma Fogh, J. and Trempe, G., New Human Tumor CellLines In: J. Fogh (ed.) Human Tumor Cells In Vitro, pp. 115-153, NewYork: Plenum Press (1975)!; BT-20, derived from primary carcinomaLasfargues, E. Y. and Ozzello L., J. Natl. Cancer Inst. 21:1131-1147(1958)! and MDA-MB-157, derived from pleural effusion of medullarycarcinoma Young, R. K. et al., In Vitro, 9:239-245 (1974)!. Each of thecarcinomas is tumorigenic in nude mice. Cultures of apparently normalmammary cells used included: HBL-100, epithelial cells derived from ahuman milk sample Polanowski, F. P. et al., In Vitro 12:328-336 (1976)!and HS0578 Bst, myoepithelial cells derived from normal tissue adjacentto primary carcinoma Hackett, A. J. et al., J. Natl. Cancer Inst.,58:1795-1806 (1977)!. Neither normal breast cell line is capable ofproducing tumors in nude mice. Murine mammary tumor cell line MMT 060562was obtained from the American Type Culture Collection.

Tissue specimens representing normal, benign, and malignant breastlesions, along with other tissues, were obtained from the PathologyDepartment of St. Joseph's Hospital, Buffalo, N.Y. All specimens werehistopathologically assessed.

6.2. Immunization and cell Fusion

Balb/c mice received, on days 0, 7 and 14, intraperitoneal injections(5×10⁶ cells per 0.2 ml) of live breast cancer cells which weresuspended in Dulbecco's phosphate-buffered saline (D-PBS). On day 60,mice received an intravenous challenge of 2×10⁶ cells in D-PBS. Cellfusion was carried out 3 days later according to the procedure developedby Kohler and Milstein (Nature (Lond.) 256:495-497 (1975)!, withmodifications: Balb/c mouse splenocytes (1×10⁸ cells) were fused in0.15M HEPES (hydroxy-ethylpiperazine-ethanesulfonic acid) buffer (pH7.5) comprising 40% (v/v) polyethylene glycol (PEG, MW=3,400) and 10%(v/v) dimethyl sulfoxide (DMSO) with 5×107 mouse myeloma cells(P3x63Ag8.653). Fused cells were distributed to 96-well culture dishesand cultured in selective hypoxanthine-aminopterin-thymidine HAT) mediumat 37° C. with 7.5% CO₂ humid atmosphere. Two to three weeks later,supernatants in a were assayed for direct binding activity on breastcancer cell lines. Hybridomas were detected in over 3,000 culture wells,with approximately 5% secreting antibodies reactive against MCF-7 breastcancer cells as determined by cell-surface radioimmunoassay (see Section6.5.). Cultures showing specificity towards breast cancer were cloned bylimiting dilution and subcloned in agarose See e.g., Schreier, M. etal., Hybridoma Techniques, pp. 11-15, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1980)!. Stable cultures of antibody-producinghybridomas were expanded in complete media RPMI-1640 media supplementedwith 10% (w/v) heat-inactivated fetal bovine serum, 100 U/ml penicillin,100 μg/ml streptomycin, and 10 μg/ml insulin (GIBCO Grand Island, N.Y.)!and exhausted culture fluids were the only source of antibodies used.

Following this procedure, hybridoma cell line F36/22, derived fromimmunization with MCF-7 cells, was obtained. The hybridoma was grown inmass culture in complete medium to produce monoclonal antibody forcharacterization studies and other applications described below.

6.3. Isotyping of F36/22 Monoclonal Antibodies

To determine the class of immunoglobulin produced by the

F36/22 hybrid, 10⁷ F36/22 hybridoma cells were washed in D-PBS,collected by centrifugation and lysed in 100 μl of 0.5% Nonidet P-40 for20 minutes on ice. Ten μl of the 40,000 ×g supernatant were examined byimmunodiffusion against antisera specific for each of murineimmunoglobulin classes: IgM, IgGl, IgG2a, IgG2b and IgG3 (MilesLaboratories, Elkhart, IN). Isotype analysis revealed that monoclonalantibody produced by F36/22 (hereinafter McAb F36/22 or monoclonalantibody F36/22) was an immunoglobulin of the γ3 class.

6.4. Immunoglobulin Preparations

The monoclonal antibody F36/22 and control murine γ3 immunoglobulin wereisolated in an identical manner using Staphylococcal aureus proteinA-Sepharose (Pharmacia, Piscataway, N.J.) as described Ey, P. L. et al.,Immunochem., 15:429-36 (1978)!. Diluted serum or ascites or clarifiedculture fluid were applied to the protein A-Sepharose adsorbent followedby removal of non-binding components with pH 6.0 buffer. Immunoglobulinof the γ3 subclass was eluted at pH 4.5, neutralized with sodiumhydroxide (NaOH) and dialyzed into PBS (phosphate buffered saline; 10 mMphosphate, pH 7.4). All immunoglobulin preparations were adjusted to 1mg/ml and frozen in small aliquots until needed.

Immunoglobulin preparations used for the present studies were at least95% pure as judged by polyacrylamide gel electrophoresis Laemmli, U. K.,Nature 227: 680-85 (1970)!.

6.5. Cell-Surface Radioimmunoassay (CS-RIA)

Unless otherwise indicated, target cells were fixed in 2%p-formaldehyde/D-PBS for 10 minutes at room temperature and stored inthe presence of assay buffer D-PBS/0.1% gelatin/1 mM PMSF (phenyl methylsulfonyl fluoride)/0.05% azide! at 4° C. for up to 2 weeks. To performthe assay, 1×10⁵ cells were incubated with 100 μl of culture fluid froma culture of the F36/22 hybridoma for 2 hours at room temperature. Cellswere washed 3 times with assay buffer before the addition of rabbitanti-mouse immunoglobulin diluted 1/500 in assay buffer Brown, J. P. etal., J. Immunol. Methods 31: 201-209 (1979)!. After 1 hour at roomtemperature, cells were washed and treated with ¹²⁵ I-labelledStaphylococcal Protein-A (Pharmacia, Piscataway, N.J.) at 2.5×10⁵ cpmper dose. After 1/2 hour, the cells were washed, dissolved in 2N NaOHand transferred to tubes, where bound radioactivity was measured.

6.6. In Vitro Characterization of Monoclonal Antibody F36/22 6.6.1.Dection and Binding of

McAb F36/22 to cultured Cells

McAb F36/22 was tested by CS-RIA against a panel of cell types (TABLEI). McAb F36/22 produced a strong reaction (greater than 10,000 cpm)against breast cancer cells MCF-7 and BT-20 and a significant reactionversus the other tested breast cancer lines. This-antibody weakly boundto a colon carcinoma and to a pancreas carcinoma (10 to 20% of maximumbinding relative to MCF-7 cells). Other cells tested, includinglymphoid/leukemoid cells, fibroblasts, and erythrocytes and culturesrepresenting apparently normal mammary epithelial cells Polanowski, F.P. et al. (1976), supra! and myoepithelial cells Hackett, A. J. et al.(1977), supra! also failed to bind significant levels of antibody F36/22(less than 10% of maximum binding). The results obtained from the cellsurface binding assay, as summarized in TABLE I, indicate that thespecificity recognized by McAb F36/22 is expressed maximally on selectedbreast cancer cells.

                  TABLE I    ______________________________________    CELL-SURFACE BINDING OF ANTIBODY F36/22.sup.a                         % of maximum                         binding activity.sup.b    Target Cell          F36/22    ______________________________________    Breast Carcinoma    BT-20                99    SK-BR-3              23    MCF-7                100    MDA-MB-157           44    Mouse Mammary Tumor MMT 060562                         5    Pancreas Carcinoma    BxPC-3               8    PANC-1               13    Prostate Carcinoma    LNCaP                3    PC-3                 6    Colon Carcinoma    HT-29                16    Lung Carcinoma    PC3                  4    CHAGO                5    Rhabdomyosarcoma    A-204                2    Melanoma    Palarmo              3    Thyroid Carcinoma    TT-4                 1    Lymphoblastoid/Leukemoid    RAJI (B-Cell)        6    DAUDI (B-Cell)       8    BALM-3 (B-Cell)      5    RPMI-6410 (B-Cell)   9    PEER (T-Cell)        5    MOLT-4 (T-Cell)      4    U-937 (monocytoid)   9    K-562 (erythroleukemoid)                         10    Normal Breast    HBL-100 (epithelial) 2    HSO578 Bst (myoepithelial)                         2    Erythrocytes    Type A               3    Type B               5    Type O               4    Type O (neuraminidase-digested).sup.c                         3    Fibroblasts    BG-9                 8    Peripheral Blood Lymphocytes                         3    ______________________________________     a. Antibody binding activity was assessed by the cellsurface     radioimmunoassay (CSRIA).     b. Maximum binding (100%) represents the cell line binding the most     antibody, and binding to other cells was expressed relative to that level     The maximum cpm (mean of 2 experiments) was as follows: antibody F36/22;     10,743 cpm.     c. Neuraminidase Digestion: Washed, type "O" erythrocytes were treated     with C. perfringens neuraminidase (BoehringerMannheim, Indianapolis, IN)     at 1U enzyme/ml of 0.1M acetate/0.15M NaCl, pH 5.5. After 1 hour at     37° C., the cells were washed with CSRIA assay buffer and assayed     for antibody binding activity as compared to acetate buffertreated contro     erythrocytes.

6.6.2. Quantitative Adsorption of McAb F36/22 to Cultured Cells

Adsorption tests were performed to determine if low binding levelsobtained from the direct-binding assays presented in Section 6.6.1. werereflecting low levels of antigen, rather than absence of antigen. Asshown previously Dippold, W. G. et al., Proc. Natl. Acad. Sci. U.S.A.,77: 6114-6118 (1980)!, target cells tested against certain monoclonalantibodies may be direct-binding-testnegative/adsorption-test-positive.The number of cells required to adsorb 50% of antibody activity wascalculated and presented as an AD₅₀ value according to the followingprocedure.

Suspensions containing different numbers of human cells (10³ to greaterthan 10⁷ cells/ml) were added to equal volumes (total volume=200 μl) ofhybridoma F36/22 culture supernatant fluid (approximately 38 ng murineIgG) and incubated for 1 hour on ice. The human cells used were: breastcarcinoma BT-20, MCF-7, and MDA-MB-157; pancreas carcinoma PANC-1; coloncarcinoma HT-29; lymphoblastoid RAJI; normal breast (epithelial)HBL-100; fibroblast BG-9; and red blood cells. After centrifugation, thebinding activity remaining in the supernatant was measured using theCS-RIA.

Appropriate dilutions of hybridoma culture supernatant fluid wereestablished by titration analysis of each antibody against breast cancertarget cells. A final dilution of antibody was chosen which was midwaydown the slope, representing 50% of maximum binding activity. Thiscorresponded to 38 ng antibody/ml final dilution for F36/22.

From the cpm of ¹²⁵ I! Protein A bound to target cells, the estimatednumber of adsorbing cells required to remove 50% of the antibodyactivity was interpolated from a regression line of best fit.

The results of adsorption analyses indicated that the capacity to adsorbMcAb F36/22 of the various cell types tested was greatest in selectedbreast cancer cells. Cultures of breast carcinoma cells BT-20, MCF-7 andMDA-MB-157 were able to adsorb F36/22 activity at AD₅₀ values of 3×10⁵,5×10⁵, and 2×10⁷, respectively. Fibroblasts and erythrocytes, along withlymphoblastoid, and colon carcinoma cells, showed no ability to adsorbMcAb F36/22. Pancreas carcinoma cells demonstrated an AD₅₀ value of1.6×10⁸ ; this indicated very low levels of epitope F36/22,approximately 500-fold less than on BT-20 breast carcinoma.

6.6.3. Reciprocal Binding Inhibition

Reciprocal binding inhibition experiments were performed to determinewhether McAb F36/22 recognized the same, closely associated or differentantigenic determinants as antibodies to: fibronectin, human DR-antigen,α-fetoprotein, pregnancy-associated α-2-macroglobulin, carcinoembryonicantigen (CEA), class I HLA-A, B or C monomorphic determinants,β2-microglobulin or ferritin.

The procedure used to test reciprocal binding inhibition was as follows:

F36/22 hybridoma antibodies were internally labelled by the addition of5 μci of ¹⁴ C! leucine (New England Nuclear, Boston, Mass.) to culturesof 2×10⁶ cells in 3 ml of leucine-free Modified Eagle's Medium (MEM)supplemented with 5% dialyzed fetal bovine serum. After 18 hours understandard culture conditions, culture fluids were clarified bycentrifugation (20,000 ×g for 1/2 hour) and dialyzed against D-PBS/1 mMleucine/1 mM PMSF/0.05% azide. For competitive analysis, 50 μl ofunlabelled antibodies (50-fold diluted sera or ascites) or 50 μl CS-RIAassay buffer (control) were allowed to react with target cells for 1hour prior to the addition of ¹⁴ C-labelled antibodies (50 μl). After 3hours the cells were washed 4 times with CS-RIA assay buffer andcounted. The 100% binding level was 2,200 cpm. Antibodies used for thesestudies included: anti-HLA monoclonal antibody W6/32 (Accurate ChemicalCorp., Hicksville, N.Y); anti-DR monoclonal antibody MAS-019 (AccurateChemical Corp.) ; anti-CEA polyclonal antiserum Chu, T. M. and Nemoto,T., J. Natl. Cancer Inst. 51:1119-1122 (1973)!; anti-α-fetoproteinpolyclonal antiserum (obtained from H. Hirai, Hokkaido University,Japan); anti-pregnancy-associated macroglobulin (obtained from E. J.Sarcione, Roswell Park Memorial Institute); anti-fibronectin monoclonalantibody MAS-037 (Accurate Chemical Corp.); anti-β2-microglobulinpolyclonal antiserum (Accurate Chemical Corp.) and antiserum to humanferritin (obtained from E. J. Sarcione, Roswell Park MemorialInstitute).

The per cent inhibition of McAb F36/22 binding activity by anti-CEAantibodies, anti-α-fetoprotein antibodies, and anti-β2-microglobulinantibodies was approximately 5% or less. The per cent inhibition byanti-fibronectin antibodies, anti-HLA antibodies, anti-DR-antigenantibodies, anti-pregnancy-associated macroglobulin antibodies, andanti-human ferritin antibodies was approximately 12% or less.

6.6.4. In Vitro Cytotoxicity of McAb F36/22

Complement-mediated cytotoxicity of monoclonal antibody was measuredusing the procedure of Brown et al. J. Immunol. Methods, 30: 23-35(1979)!. Cells were incubated in the presence of McAb F36/22 plus rabbitcomplement for 1.5 hours after which time cells were evaluated for theirability to metabolically incorporate radioactive leucine as compared tountreated controls. Cells examined included Chago lung carcinoma Rabson,A. S. et al., J. Natl. Cancer Inst., 50: 660-74 (1973)!, Daudi lymphomaKlein, E. et al., Cancer Res., 28:1300-10 (1968)! and MCF-7 breastcarcinoma Soule, H. D. et al., J. Natl. Cancer Inst., 51: 1409-16(1973)!.

Titration data summarized in TABLE II demonstrate that McAb F36/22 iscytotoxic to MCF-7 breast carcinoma cells in the presence of complement.This cytotoxic effect has been demonstrated at high dilutions ofantibody. Also, the cytotoxicity observed is specific forantigen-expressing tumor cells. Cell lines expressing undetectableamounts of determinant, such as Chago lung carcinoma and Daudi lymphoma,were not significantly killed in the presence of complement plus McAbF36/22 at high dilutions which were cytotoxic to breast carcinoma.

                  TABLE II    ______________________________________    IN VITRO CYTOTOXICITY OF McAb F36/22    Approx. % Cytotoxicity at Various Antibody Dilutions            Dilutions    Cell Line 10.sup.0                    10.sup.-1                             10.sup.-2                                  10.sup.-3                                         10.sup.-4                                              10.sup.-5    ______________________________________    MCF-7     65    60       65   25     10   0    Chago     55    0        0    0      0    0    Daudi     60    0        0    0      0    0    ______________________________________

In vitro antibody mediated complement cytotoxicity assays were repeatedon BT-20 and MCF-7 cell lines. The results showed specific cell lysis(greater than 50% cell kill) at a concentration of 1 ng of McAb F36/22per ml of medium and no cytolysis of 11 different non-breast cell lines(8 leukemia, 2 carcinomas, 1 lymphoma) at a concentration of 2.5 μg/ml.Rabbit or human complement at 1:10 dilution were both shown to beequally effective. Antibody without complement gave no significantcytolysis.

The rapidity of the tumoricidal process (see Section 6.9.3.) and the invitro indication of specific cytolysis at very low concentration of McAbF36/22 with complement point to the involvement of a complement mediatedeffect. It has been reported that murine γ3 antibodies bind with lowaffinity and low frequency to murine macrophages Ralph, P. et al., J.Immunol 125:1885-1888 (1980)!. The possibility exists, however, thatantibody mediated cellular cytotoxicity also takes place.

6.6.5. Reactivity of McAb F36/22 with Estrophilin Complexes

Experiments were performed to define the possible role of estrophilin inthe reaction of McAb F36/22 against human breast carcinomas. Using animmunochemical assay, McAb F36/22 added prior to labelled estradiol wasunable to competitively inhibit the formation of estradiol-estrophilincomplexes. However, the possibility existed that even through F36/22 didnot react with an epitope involved in the hormone-receptor binding site,it may detect an epitope located elsewhere on the estrophilin molecule.To evaluate this, the following procedure was used.

Radiolabelled estradiol-estrophilin complexes were prepared using MCF-7cytosol as described by Horwitz and McGuire Cancer Res. 37: 1733-1738(1977)!, except that ¹²⁵ I-labelled estradiol was used (New EnglandNuclear, 2000 μci/mM) in place of the tritiated reagent. Preformedcomplexes were incubated at 4° C. for 1 hour with McAb F36/22 (25 μg) orcontrol immunoglobulin (25 μg) from serum of a non-immunized mouse asdescribed previously Greene, G. L. et al., Proc. Natl. Acad. Sci. U.S.A.77:5115-5119 (1980)!. Each mixture was individually applied to a column(1×45 cm) packed with S-200 chromatography media and equilibrated withrunning buffer (10 mM Tris-HCl, pH 7.4 containing 1.5 mM DTT(dithiothreitol), 1 mM EDTA (ethylenediaminetetraacetic acid) and 400 mMKCl). Successive 1 ml fractions were collected and radioactivity wasmeasured. Purified human hemoglobin (Sigma) was used as an internalmarker of the 4S protein peak.

The estradiol-estrophilin complexes eluted in the 4S region both in thepresence and absence of McAb F36/22 as compared to monoclonal antibodieswhich react with estrophilin Greene, G. L. et al. (1980), supra!, thusindicating no reactivity with the estrophilin molecule.

6.6.6. Relation between Estradiol Concentration and McAb F36/22 BindingLevels

Hormonal involvement of antigen expression in MCF-7 breast cancer wasinvestigated. A study was performed to ascertain if increased antigenexpression of estrogen receptor-rich (ER-rich) tumors was related to theestradiol concentration.

First, endogenous estradiol was removed from fetal bovine serum (FBS)used in normal complete cell culture medium. The basic procedure usedwas reported earlier by Westley and Rochefort Cell 20:353-362 (1980)!.Fifty ml of FBS #309 (GIBCO) was depleted of estrogen as follows: A DCCsuspension (0.025% Norit A and 0.0025% dextran in TESH (10 mM Tris-HCl,pH 7.4, containing 1 mM EDTA and 1.5 mM DDT buffer) was preparedaccording to the method of Garola and McGuire Cancer Res. 38:2216-2220(1978)!. The DCC (250 mg charcoal, 25 mg of dextran per 50 ml of FBS)was added to the FBS and mixed on an aliquot shaker at 55° C. for 30minutes. After 2 treatments with DCC, the steroid-depleted FBS wassterile filtered through 0.2 μm Nalgene filters. Complete mediumconsisted of RPMI-1640 containing 10% estrogen-depleted FBS, 100 U/mlpenicillin and 100 μg/ml streptomycin. All tissue culture reagents wereobtained from GIBCO, Grand Island, N.Y.

The following protocol was used to determine the effect of exogenousestradiol on antibody binding levels. MCF-7 cells were washed twice inD-PBS, resuspended in steroid-depleted medium, and lightly seeded intoreplicate flasks. The medium was replaced every third day. On day 10,various final concentrations of estradiol-17 β-cypionate (Sigma) (0,10⁻³, 10⁻⁶, 10⁻⁹ M) in DCC-treated medium were added to the flasks.After 72 hours of stimulation, the cells were harvested by gentleaspiration, counted using a hemocytometer and aliquoted. Cells werewashed twice in D-PBS and then fixed for 10 minutes in 2% p-formaldehydein 30 mM phosphate buffer, pH 7.2. The cells were again washed andtreated with 0.5% saponin in 30 mM phosphate buffer, pH-7.2, for 3minutes to reveal intracellular binding sites Fambrough, D. M. andDevreotes, P. N., J. Cell. Biol., 76: 237-244 (1978)!. The cells werefurther washed and incubated with ¹²⁵ I!-F(ab')₂ fragments of McAbF36/22 for 60 minutes (200,000 cpm/tube), washed 4 times, and the countswere measured in a Packard gamma spectrometer.

Competitive inhibition, using saturating doses (10 μg/sample) ofunlabelled McAb F36/22 prior to the addition of the labeled F(ab')₂fragments permitted an estimation of non-specific background. Inaddition, an irrelevant IgG3 control antibody (10 μg/sample) wasincubated prior to the labelled fragments of McAb F36/22, in order toassess the specificity of the binding reaction.

Using these in vitro conditions, exogenous estradiol concentrations of10⁻³ and 10⁻⁶ M were shown to significantly increase the amount ofantibody binding levels as compared to control-treated cultures (p lessthan 0.001) (TABLE III). Fixation of the MCF-7 cells with p-formaldehydealone failed to elicit any significant changes in cell-surface bindingin response to increasing concentrations of estradiol. However,treatment of the cells with saponin, which reveals intra-cytoplasmicbinding sites, greatly promoted the binding of McAb F36/22. This invitro observation coincides with the in vivo tumor staining patterns(see Section 6.9.), where the predominant location of immunoperoxidasestaining in ER-rich tumors is cytoplasmic (80%).

                  TABLE III    ______________________________________    RELATIONSHIP BETWEEN ESTRADIOL LEVELS    AND ANTIGEN EXPRESSION OF MCF-7 CELLS.sup.a                      .sup.125 I!-F(ab').sub.2 cpm bound/    Final Estradiol Concentration                     10.sup.5 MCF-7 Cells.sup.b    ______________________________________    0                3975 ± 556    10.sup.-9 M      4213 ± 842.sup.c    10.sup.-6 M      5333 ± 640.sup.d    10.sup.-3 M      6133 ± 306.sup.d    ______________________________________     a. MCF7 cells, maintained in estrogendepleted medium for 10 days, were     stimulated with various final concentrations of estradiol. After 72 hours     the cells were harvested, counted and aliquoted into 3 tubes/flask. The     cells were fixed in buffered 2% pformaldehyde for 10 minutes, and further     treated with 0.5% bufferedsaponin for 3 minutes to reveal intracellular     binding sites. The cells were then incubated with  .sup.125 IF(ab').sub.2     fragments of McAb F36/22 for 60 minutes (200,000 cpm/tube), and the count     bound were measured in a gamma spectrometer.     b. In this experiment, specific binding of McAb F36/22 fragments was     calculated by subtracting the nonspecific binding in the presence of     saturating levels of unlabelled McAb F36/22 from the total counts bound.     Results were expressed as  .sup.125 IF(ab').sub.2 cpm bound/10.sup.5 MCF7     cells.     c. Not statistically significant.     d. Statistically significant antibody binding (p less than 0.05) as     compared to control treated cultures (Student's t test).

Because exogenous estradiol was capable of increasing the cytoplasmicbinding of McAb F36/22 to saponin-treated MCF cells, the antigen whichMcAb F36/22 recognizes may represent a protein whose sythesis isregulated under the estrogen-response machinery of the breast cancercell. The existence of several estrogen-regulated proteins has been wellestablished see e.g., Butler, W. B. et al., Biochem. Biophys. Res.Commun., 90:1328-1334 (1979); and Capany, F. et al., Biochem. Biophys.Res. Commun., 108:8-15 (1982)! and monoclonal antibodies toestrogen-receptor status-related components have been described Ciocca,D. R. et al., Cancer Res., 42: 4256-4258 (1982)!. Identification of suchproteins may add new information regarding hormonally-dependentregulatory controls involved in breast cancer which may ultimately leadto new therapies.

6.7. Characterization of the Antigen Recognized by Monoclonal AntibodyF36/22 6.7.1. Solid-Phase Adsorption of BT-20 Breast Carcinoma Antigenby Lectins

The following solid-phase ligands were tested for their capacity toreact with antigen occurring in BT-20 breast cancer cell lysates:Concanavalin A-Sepharose CL-4B (Pharmacia), in 50 mM sodium acetate/1MNaCl/1 mM CaCl₂ /1 mM MnCl₂, pH 6.0 (binding capacity 8.5 mg/ml gel);Wheat-germ Sepharose 6MB (Pharmacia), in D-PBS (binding capacity 5mg/ml; Peanut Agglutinin-Ultrogel (LKB, Rockville, Md.), in D-PBS(binding capacity 4 mg/ml); and Gelatin-Agarose (Bio-Rad, Richmond,Calif.), in D-PBS (binding capacity 6 mg/ml).

Samples of BT-20 breast cancer cell lysates (8 mg protein/ml) wereprepared in D-PBS/0.5% sodium deoxycholate and were incubated with anequal volume of solid-phase ligand (25% v/v suspension in theappropriate buffer). After 1 hour at 4° C., the beads were washed 3times and incubated with F36/22 hybridoma culture fluid for 2 hours at4° C. Subsequently, antibody binding activity was revealed using theCS-RIA (Section 6.5.) procedure as for target cells. Since standardpreparations of purified BT-20 breast cancer antigen was not availablein this set of experiments as reference material, results of theseassays were used to obtain qualitative information regarding lectinbinding activities.

Concanavalin A was able to bind soluble components from BT-20 breastcancer cell lysates which in turn exhibited the capacity to adsorb McAbF36/22. Peanut agglutinin and wheat germ lectins had a more limitedability to bind F36/22 antibody-reactive components from BT-20 celllysates (less than 20% of that exhibited by concanavalin). Also testedwas insolubilized gelatin, which produced no antibody binding activityafter treatment with breast cancer cell lysates.

6.7.2. Antigen Modulation

The stability of cell surface antigen after treatment of MCF-7 cellswith McAb F36/22 was investigated.

To sensitive viable MCF-7 breast carcinoma cells growing in microwellplates, the cells were allowed to incubate in the presence of saturatingdoses of monoclonal antibody (10 μg/ml of sterile complete medium). Theexperiment was run at 0°, 23° and 37° C. At successive time intervals upto 4 hours the cells were washed and assayed for McAb F36/22 binding asdescribed in Section 6.5. Additionally, monoclonal antibody-sensitizedcells were also examined under immunofluorescence using fluorescentlabelled antibody (where the fluorescent label was fluoresceinisothiocyanate (FITC)) against murine immunoglobulin (Accurate ChemicalCo. Hicksville, N.Y.) see Papsidero, L. D. et al. Hybridoma, 1:275-82(1982).

No loss of McAb F36/22 binding levels was noted after 4 hours even atthe highest temperature tested, 37° C. This indicated no externalmodulation or redistribution of McAb F36/22-complexed cell surfaceantigen. Immunofluorescence examination of identically-treated cellsshowed the presence of strong staining which was limited to the cellsurface.

6.7.3. Release of Antigen Recognized by McAb F36/22 by Tumor Cells

To examine if antigen recognized by McAb F36/22 was spontaneouslyreleased by tumor cells, pleural fluids were examined using anitrocellulose direct-binding assay procedure. These fluids wereobtained from patients with disseminated breast carcinoma and werehistopathologically evaluated for the presence of tumor cells.

Detection of antigen which was adsorbed to nitrocellulose paper wasperformed using an antigen spot test as described by Herbrink et al. J.Immunol. Methods 48:293-8 (1982)!. Briefly, antigen samples in the formof pleural fluids (or chromatography fractions as described in Section6.7.4.) were applied as 10 μl drops to nitrocellulose transfer paper(BioRad, Richmond, Calif.) and allowed to dry for 15 minutes Thereafterthe paper was inactivated by incubation in assay buffer (0.1M Tris-HCl,pH 7.4 containing 0.25% (w/v) gelatin and 0.5% (v/v) nonidet P40). Thepaper was then incubated for 18 hours at 4° C. with antibody F36/22 at10 μg/ml of complete culture medium. After washing the paper 4 timeswith assay buffer, the bound antibody was detected by incubating thepaper with ¹²⁵ I-labelled goat F(ab')₂ antibodies to murineimmunoglobulin Fraker, P. J. and Speck, Jr., J. C., Biochem. Biophys.Res. Commun., 80:849-57 (1978). After washing, the paper was sectionedand individual samples counted for bound radioactivity.

To avoid spurious binding to human immunoglobulins, the radiolabelledanti-murine antibody was affinity-purified on murineimmunoglobulin-Sepharose columns and further adsorbed with solid-phasehuman immunoglobulins March, S. C. et al., Anal. Biochem. 60:149-52(1974)1. The resulting antibody preparation demonstrated less than 1%cross-reactivity against purified human IgG.

When compared to normal serum donors, the malignant effusions testedexhibited an increased level of McAb F36/22 binding activity andincreased incidence of elevated antigen amounts (p less than 0.02); 9out of 10 samples derived from healthy blood serum donors bound lessthan or equal to 3923 cpm while 17 out of 24 pleural fluid samples frombreast carcinoma patients exhibited binding of 3923 cpm or more, withone sample binding as high as 1×10⁵ cpm. Also, when control murineimmunoglobulin was substituted for McAb F36/22, no difference wasobserved between normal donors and cancer patients, indicatingimmunological specificity for the reaction.

Though physicochemical characterization of the pleural effusion-borneantigen(s) carrying the specific epitope recognized by McAb F36/22 werenot performed as in Section 6.7.4., the results of the antigen spot testsuggest serological applications for McAb F36/22 as described below.

6.7.4. Gel Filtration Chromatography of MCF-7 Antigen Recognized by McAbF36/22

MCF-7 primary breast carcinoma cells were homogenized at 4° C. indetergent buffer (10 mM Tris, pH 7.4/0.5% (w/v) sodium deoxycholate/0.5mM phenylmethylsulfonyl fluoride). Nuclei and debris were removed bycentrifugation at 40,000×g for 1 hour. Five ml of the clear supernatantwere applied to a column (2.6×100 cm) packed with Sephacryl S-300 gelfiltration media (Pharmacia) and equilibrated with the above detergentbuffer. The column was run at a flow rate of 15 ml/hr and fractions of 5ml were collected. The column was pre-calibrated using protein markersof known molecular weight thyroglobulin (19.2s), immunoglobulin G (7s),and serum albumin (4.5s)!.

Fractions collected were individually tested for their immunoreactivityagainst monoclonal antibody F36/22 using the antigen spot test (seeSection 6.7.3.). As control, the test was also performed using P3X63-Ag8immunoglobulin as a substitute for specific antibody. Fractions elutingat approximately 5s demonstrated significant reactivity against McAbF36/22. These fractions showed no such reactivity versus controlimmunoglobulin.

6.7.5. Effect of Enzyme Digestions on Cell Surface Antigenicity

Breast carcinoma cells (MCF-7) at a concentration of 1×10⁶ cells/ml weresubjected to the following reagents (final concentration) at 37° C.:Papain (Boehringer-Mannheim, Indianapolis, Ind.) at 0.8 U/ml in pH 6.3PBS containing 1 mM cysteine and 1 mM EDTA; Trypsin (GIBCO) at 1372 U/mlof Hank's salt solution containing 0.5 mM EDTA; Pepsin (Sigma, St.Louis, Mich.) at 243 U/ml of acetate buffer pH 4.5; Neuraminidase(Boehringer-Mannheim) at 0.05 U/ml of acetate buffer pH 5.0 containing 1mM phenylmethyl sulfonyl fluoride; α-mannosidase (Sigma) at 1 U/ml ofacetate buffer, pH 4.5 containing 1 mM ZnCl; Sodium-meta-periodate(Sigma) at 50 mM in PBS.

Cells were incubated with proteolytic enzymes for 5 minutes while theincubations with carbohydrate-cleaving agents were allowed to continuefor 30 minutes. Cell viability was greater than 85% after each of thesetreatments. Subsequently, the cells were washed and assayed for residualantigenicity by direct-binding CS-RIA as described in Section 6.5. usingmonoclonal antibody F36/22.

The cell surface component recognized by F36/22 was labile underproteolytic conditions and resisted carbohydrate-cleaving reagents.

Direct binding of McAb F36/22 to breast carcinoma cells, and hence,cell-surface antigenicity was significantly diminished after treatmentof the cells with papain and trypsin. Papain digestion and trypsindigestion reduced direct binding activity to approximately 40% and 10%of control values, respectively. Treatment with pepsin showed no effecton direct binding. All carbohydrate-cleaving agents tested produced adramatic increase in antibody binding activity: Neuraminidase,α-mannosidase and periodate increased direct binding by 200%, 250% and400% of control values, respectively. This effect was not based uponcellular uptake of the antibody reagents, as in each case greater than85% viability of the cells was observed post-treatment.

The reason for the dramatic increase in antigenicity following treatmentwith carbohydrate-active reagents such as periodate and glycosidases maybe that these reagents clear and/or "unmask" the cell surface of somecarbohydrate moieties thus allowing greater access of McAb F36/22 todeterminants directly adjacent to the tell membrane.

6.8. Dection of Ductal Carcinoma Antigen in Breast Cancer Sera usingMonoclonal Antibody F36/22

A quantitative immunoassay procedure was constructed to evaluate levelsof the ductal carcinoma antigen (DCA) recognized by McAb F36/22 in ahuman fluid sample. The cell-surface component(s) is associated with aline of epithelial tumors of ductular lineage.

6.8.1. Enzyme Immunoassay Procedure

The sandwich type of enzyme immunoassay was developed based uponobservations presented below (Sec. 6.9.4. and 6.9.5.) which indicatedthe occurrence of multiple antibody combining sites on DCA.

To develop the procedure, antigen standards comprising a papain digestof breast tumor specimens (shown to contain the antigen byimmunoperoxidase staining) were used. Human primary breast cancerspecimens were pooled and homogenized in 10 volumes of 10 mM Trisbuffer, pH 7.4, containing 0.2% (w/v) sodium deoxycholate at 4° C. Thehomogenate was quickly brought to 37° C. and the following reagents(final concentration) were added while stirring: 1 mM cysteine (Sigma),1 mM EDTA (Sigma), and papain (0.8 unit/ml) (Boehringer-Mannheim,Indianapolis, Ind.). After 5 minutes digestion was stopped by theaddition of 5 mM iodoacetamide (Sigma). The homogenate was centrifugedat 100,000×g for 1 hour at 40° C., then extensively dialyzed against 10mM Tris/0.9% NaCl solution buffer, pH 7.4, containingphenylmethysulfonyl fluoride and aminocaproic acid, each at 10 mM. Thehomogenate was frozen in small aliquots at a concentration of 0.5 mg ofprotein/ml. Polyacrylamide gel electrophoresis of the papain digestrevealed the presence of several major glycoproteins.

The dose response curve generated for the immunoassay proceduremeasuring DCA demonstrated linearty (r=0.98, linear regression analysis)between antigen input of 0.625 to 10 units/ml. For serum analysis, therange was 16.25 to 260 units/ml, since these samples were diluted26-fold prior to assay. The values of antigen content were arbitrarilyassigned in the absence of purified antigen during development of theassay.

Solid-phase preparations of McAbs were prepared using CNBr-activatedSepharose (Pharmacia). Microtiter plates (Nunc I Immunoplates; GrandIsland Biological Co., Grand Island, N.Y.) were coated with McAbs (200μl/well) in 50 mM carbonate-bicarbonate buffer, pH 9.6, for 18 hours at4° C. After removal of the antibody solution, residual protein bindingsites on the plastic were blocked by the addition of 200 μl of assaybuffer PBS containing 1% (v/v) murine serum and 1% (w/v) bovinealbumin!. After 1 hour of incubation at room temperature, the coatedplates were used immediately for the assay procedure.

To perform the assay, 200 μl (10 units/ml) samples, diluted in assaybuffer, were applied for 1.5 hours at 37° C. After 3 washes using assaybuffer, 200 μl of McAb F36/22 covalently conjugated to horseradishperoxidase (Sigma, Type VI) was applied to each well for 1.5 hours at37° C. The conjugate was diluted to a concentration of 0.5 μg ofimmunoglobulin per ml of PBS containing 10% (v/v) murine serum.Following a wash procedure as above, 200 μl of substrate per well wereapplied for 0.5 hours at room temperature. Substrate solution contained0.4 mg of o-phenylenediamine per ml of pH 5.0 citrate buffer and 0.003%hydrogen peroxide. The reaction was stopped by addition of 50 μl of 2Nsulfuric acid, and absorbance was monitored at 488 nM using an enzymeassay plate reader (Fisher Scientific Co., Pittsburgh, Pa).

The percentage of bound enzyme conjugate was calculated by the formula:

    (B--B.sub.0)(B.sub.t -B.sub.0)×100

where B=absorbance of the sample, B_(t) =maximal absorbance, and B₀=absorbance of the blank. Each assay was performed in triplicate using astandard papain digest and 26-fold diluted serum samples diluted inassay buffer. Samples producing an absorbance corresponding to greaterthan 50% bound were serially diluted and reassayed. Samples showing astandard deviation above 10% were also reassayed. An interassaycoefficient of variation of 12% was observed for 14 consecutive assays.

Specificity of the immunoassay was examined by substituting variousantibody reagents at the solid phase, including McAb F5 (prostateantigen specific Papsidero, Hybridoma 2:139-147 (1983)!; McAb M7/105,distinct tumor antigen specific (Papsidero et al., Cancer Res. 43:1741-1747 (1983)); and nonimmune murine serum. Of the solid phaseantibodies only antibody F36/22 bound antigen at high dilutions.

Levels of serum ductal carcinoma antigen were detected for normalcontrols subjects, patients with benign and malignant breast diseasesand patients with prostatic and gastrointestinal cancer.

Sera obtained from 64 apparently healthy individuals exhibited a meanvalue of approximately 28 units of DCA/ml. Only 3% of the samplesexpressed serum antigen at 70 units/ml or above, and this value wasarbitrarily chosen as cutoff for elevated serum levels. This group, inaddition to laboratory personnel, contained 32 samples obtained fromage-matched controls (Table IV). No statistically significant differencewas observed between these groups regarding circulating DCA levels.Furthermore, long-term (1 year or greater) storage of sera or 3 cyclesof freeze-thaw failed to significantly alter DCA values. Similar valueswere obtained using freshly drawn and unfrozen sera from 10 volunteers(mean value, 29 units/ml). All sera to be evaluated were diluted 26-foldprior to assay, as based upon antigen recovery experiments. At dilutionsbelow 20-fold, the recovery of antigenic activity was less than 100% anda source of experimental underestimate of DCA levels.

Sera from patients with benign disease of the breast (most withfibrocystic disease) exhibited a mean DCA value of 41.5 units/ml (TableIV). The incidence of values above 70 units/ml was 13% (5 of 40specimens). Patients with breast cancer (with evidence of disease)exhibited a wide range of circulating levels of DCA with a mean valueabove 700 units/ml. The incidence of elevated values for this group wasapproximately 53% (61 out of 116 specimens) Patients with early stagedisease or with no clinical evidence of disease demonstrated a decreasedincidence of elevated serum antigen values (Table IV). Approximately 30%of these specimens contained levels of DCA above 70 units/ml.

                  TABLE IV    ______________________________________    DETECTION OF DUCTAL CARCINOMA ANTIGEN IN SERUM                  Antigen level (units/ml)                    No.                 >70 units/    Group           tested Mean         ml (%)    ______________________________________    Apparently healthy controls    Lab personnel   32      28.9 ± 18.2.sup.a                                        1 (3)    Age-matched     32     27.9 ± 16.3                                        1 (3)    Patients with benign breast                    40     41.5 ± 43.2                                         5 (13)    disease    Patients with non-breast cancer    Prostate        65     28.3 ± 24.8                                         7 (11)    Miscellaneous gastrointestinal                    112     60.2 ± 50.4.sup.b                                        30 (27)    Patients with breast cancer    Early stage     15      49.9 ± 23.7.sup.b                                         5 (33)    Late stage (ED.sup.c)                    101     719.1 ± 3446.2.sup.b                                        56 (56)    Late stage (NED)                    15      52.9 ± 22.8.sup.b                                         4 (27)    ______________________________________     .sup.a Mean ± S.D.     .sup.b p <0.05 as compared to control population (Student's t test     analysis).     .sup.c ED, patients with clinically detectable evidence of disease; NED,     no evidence of clinically detectable disease.

Serum samples were also obtained from 12 patients prior to and 4 daysafter mastectomy in order to evaluate the response of serum antigenlevels to surgery. Of these 12 patients, 4 showed pretreatment levelsgreater than 70 units/ml. After surgery, each patient in this lattergroup demonstrated a decrease in serum DCA level to the normal range(Table V).

                  TABLE V    ______________________________________    ANALYSIS OF ANTIGEN LEVEL PRIOR TO AND AFTER    MASTECTOMY                Antigen level (units/ml)    Patient       Presurgery                           Postsurgery.sup.a    ______________________________________    361           82.7     38.4    347           73.3     19.2    283           90.7     68.9    269           71.8     43.3    ______________________________________     .sup.a Sera obtained 4 days postsurgically. In each case shown, specimens     obtained after treatment demonstrated significantly reduced antigen level     (p <0.001).

Sera obtained from 65 patients with prostatic cancer and 112 patientswith gastrointestinal cancer (advanced disease) were evaluated (TableIV). The incidence of elevated DCA values was 11 and 27%, respectively.Mean serum values from the group with gastrointestinal cancer (60units/ml) were significantly higher than control levels.

All human sera examined also showed detectable levels of antigenactivity. The source of antigen, as occurring in the circulation ofnormal individuals, is not known. Since a few normal ductal epitheliastructures have been shown to express antigen, their contribution iscirculating immunoreactivity appears probable. However, the quantitativerange of antigen levels in the normal serum controls is narrow and maybe discriminated from specimens of patients' sera.

The highest incidence and levels of circulating DCA were associated withpatients with clinical evidence of breast cancer. The incidence ofelevated serum levels in this group (approximately 50%) may be basedupon the observations described in Section 6.10. which indicated thatonly a subset of breast tumors expresses antigen. Since the use of McAbsis associated with the description of intertumoral antigenicheterogeneity, this observed incidence is not surprising. There alsoappears to be, a strong correlation between the incidence of serum DCAlevels and intratumoral expression of antigen. Tumor histotypes such asbreast cancer, which exhibit a high incidence of tumor antigen, alsoshow a related incidence of elevated circulating DCA values. These datapredict that patients with mesenchymally derived tumors may present withlow frequencies of circulating DCA.

Of clinical importance, these data also suggest that patients withovarian ductal carcinomas may exhibit a high incidence of DCA serumelevations. As shown in Table V 100% of such tumors express antigen andmay potentially be diagnosed by serodetection. In any event,differential diagnosis of ductal carcinomas, as based upon serum values,may be facilitated in a manner analogous to our studies performed usingsolid tumor specimens.

Using a limited number of mastectomy patients with primary localizeddisease, a significant decrease in serum DCA occurred postoperatively.These data indirectly indicate a relationship between serum DCA levelsand tumor load, suggesting that such measurements may be of value forpatient monitoring. Perhaps equally important, is the fact that DCAvalues in no instance were seen to increase postsurgically. Thisdirectly implies that the antigen under measurement does not represent anonspecific "acute-phase reactant", the levels of which sharply increasefollowing surgical therapy.

6.8.2. Biochemical Characteristics of Circulating Sera

Serum samples (0.5 ml) from 5 patients with breast cancer were subjectedto size fractionation. Samples were filtered over a column (1.6×90 cm)packed with Sephacryl S-400 (Pharmacia). Column fractions eluted withPBS were diluted in enzyme assay buffer (PBS containing 1% (v/v) murineserum and 1% (w/v) bovine albumin) and evaluated for antigen activity.The column was pre-calibrated using protein standards of known molecularweight, including bovine thyroglobulin, aldolase, catalase, and albumin(Sigma).

Column fractions exhibiting peak antigen activity were furtherfractionated under equilibrium density gradient ultracentrifugation.Samples in PBS were brought to a density of 1.45 g of cesium chlorideper ml. After centrifugation (1.5×10⁶ ×g for 72 hours at 10° C.),fractions were assayed for antigen activity and for density using ananalytical balance.

Fractions obtained were evaluated for immunoreactivity using the enzymeimmunoassay procedure.

Results demonstrated the presence of antigen activity eluting inhigh-molecular weight fractions ahead of the 669,000 molecular weightprotein. Results were similar among the specimens examined. The densityof the serum antigen was evaluated using equilibrium ultracentrifugationin the presence of cesium chloride and exhibited a peak at approximately1.45 g/ml, although a broad range of activity was observed.

6.8.3. Solid Phase Adsorption of Serum Antigen

The ability of serum antigen to interact with various solidphase-adsorbents also was studied. Serum specimens obtained frompatients with breast cancer were incubated with the followingsolid-phase adsorbents (Sigma): (a) concanvalin A:Sepharose; (b) wheatgerm:Sepharose; (c) peanut agglutinin; (d) lentil lectin:Sepharose; and(e) Protein A:Sepharose. Sepharose adsorbents derivatized with McAbsF36/22 and F5 were prepared using cyanogen bromide-activated Sepharoseaccording to recommendations from the manufacturer (Pharmacia).Nonderivatized Sepharose served as a negative control (See also Sec.6.9.6).

For adsorption studies, serum dilutions were incubated with adsorbents(50%, v/v) at a volume ratio of 1:40 for 18 hours at 4° C. All testswere performed in triplicate. After centrifugation (500 ×g), thesupernatant was assayed for antigen activity.

Statistical evaluations (Student's t test) indicated that thesolid-phase adsorbents prepared from wheat germ lectin and McAb F36/22bound statistically significant (p less than 0.01) amounts of serumantigen as compared to control Sepharose.

As seen in FIG. 1, the significant amount of antigen bound byimmobilized wheat germ lectin indicated the presence of availableβ-N-acetylglucosaminyl groups. The specificity of this interaction wasconfirmed by competitive inhibition using β-N-acetylglucosamine (100ng/ml). Negligible binding to Protein A: Sepharose suggested the absenceof circulating immune complexes. Significant amounts of antigen were notobserved to react with other lectins, including concanavalin A, lentillectin, and peanut agglutinin.

6.9. Immunoaffinity Isolation of Ductal Carcinoma Antigen usingMonoclonal Antibody F36/22

McAb F36/22 recognizes a mucin-like glycoprotein occurring in malignanteffusions and in human sera. Mucins or mucoproteins are a class ofglycoproteins in which acid mucopolysaccharides, usually containing twotypes of alternating monosaccharide units of which at least one has anacid group, are complexed with specific proteins. Characteristics of thepurified component appear distinct from previously described circulatingantigens of breast and ovarian cancer. The data also indicate thatmucins may carry tumor-related determinants.

Purification of antigen from the circulation of cancer patients wasachieved through the use of sequential affinity chromatography stepsbased upon antigen interaction with McAb F36/22 and wheat germ lectin.

20 6.9.1. Purification of Antigen

The antigen recognized by McAb F36/22 was purified from malignanteffusions obtained from thoracentesis of patients withhisto-pathologically confirmed breast cancer. Prior to their use, allfluids were clarified by filtration and the following proteaseinhibitors were added: 0.1M 6-aminohexanoic acid, 10 mM aprotinin and1.0 mM phenylmethyl sulfonylfluoride (final concentrations).

Malignant effusions, adjusted to pH 7.0 were loaded onto a series ofthree columns: Sepharose 4B, nonimmune murine immunoglobulin- and McAbF36/22-Sepharose 4B Pak et al., Molec. Immun. 20: 1369-1377 (1983)!. Allcolumns were pre-equilibrated with PBS containing the proteaseinhibitors described supra. After loading, the antibody adsorbent waswashed with 25-50 bed volumes of pH 7.0 phosphate buffer containing 1MNaCl. Subsequently, antigen was eluted using 0.2M citrate, pH 2.5, and 1ml fractions were collected in tubes containing 1 ml of 1M Tris buffer,pH 8.4. Eluted material was dialyzed at 4° C. against deionized waterand lyophilized to dryness.

Material obtained from the antibody adsorbent was dissolved in lectinaffinity buffer (phosphate buffer, pH 7.0 containing 0.3 M NaCl) andapplied to a column packed with 10 ml of wheat germ lectin-Sepharose(Pharmacia).

After washing the column with 25 bed volumes of running buffer, boundcomponents were eluted in the presence of N-acetyl-β-D-glucosamine (100mg/ml). This material was extensively dialyzed against deionized waterat 4° C. and lyophilized. The yield of isolated antigen was determinedusing an analytical balance and ranged from between 50 to 100 ng/ml oforiginal effusion.

6.9.2. Size Fractionation of Antigen

Malignant effusions obtained from patients with breast cancer (BCA) orovarian cancer (OCA) or purified antigen preparation were sizefractionated on columns packed either with Sephacryl S-400 or SepharoseCL-4B (Pharmacia).

A 0.5 ml sample was applied to a 1.5×100 cm chromatographic columnpacked with Sepharose CL-4B media. Fractions (2 ml) were eluted withphosphate buffer, pH 7.0, containing 1.0M NaCl and monitored for protein(absorbance at 280 nm) and antigen content (absorbance at 488 nm) usingthe enzyme immunoassay procedure described below.

Elevated levels of this ductal carcinoma antigen have also been detectedin a high percentage of malignant effusions and in the sera of selectedpatients with ductal carcinomas (See Sec. 6.8.). As shown in FIG. 2,antigen occurring in effusions from both breast and ovarian cancer wasfound in high molecular weight fractions obtained after molecular sievechromatographies. In all fluids studied, peak antigen activity elutedahead of a 669,000 molecular weight marker protein.

6.9.3. Polyacrylamide Gel Electrophoresis

After purification of antigen from effusions, ductal carcinoma antigenwas analyzed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE).

Samples (10 μg) were applied to 7.5% and 4% SDS-PAGE gels according tothe method of Laemmli (1970, Nature, Lond. 227:680-685). Isolatedmaterial did not enter 7.5% SDS-PAGE gels but was visualized as anelectrophoretically homogeneous component on 4.0% gels. This componentdid not stain with Coomassie blue R-250 protein stain, but did reactwhen stained by the periodic-acid Schiff precedure to produce an intenseband, indicating a high percentage of carbohydrate. Fairbanks,Biochemistry 10:2606-2617 (1971)!. No structural differences were notedunder non-reducing conditions.

The antigen was also analyzed by Western tansfer of purified antigenonto nitrocellulose paper. Briefly, proteins were separated by SDS-PAGEand electrophoretically-transferred to nitrocellulose using theprocedure of Towbin et al. (Proc. Natl. Acad. Sci U.S.A. 76:4350-4355(1979)!. Residual protein-binding sites on the paper were blocked byincubation with 5% (w/v) bovine serum albumin in PBS, for 1 hour at 37°C. The nitrocellulose was then incubated with McAb F36/22 (10 μg/ml) ornonimmune murine serum for 18 hours at 4° C. After three washes (10minutes each) with PBS, the paper was incubated for 1.0 hour at 37° C.with rabbit antiserum to murine immunoglobulin (Miles) diluted 500-foldin PBS. Following a wash procedure, as earlier, the nitrocellulosetransfers were allowed to react with protein A-peroxidase conjugates(Zymed Laboratories) (500-fold diluted) for 0.5 hours at 37° C. Thewashed transfers were developed in substrate solution containing 0.1 mg4-chloro-1-naphthol/ml of PBS with 0.003% hydrogen peroxide. Afterdevelopment (10-30 minutes at room temperature) the transfers werewashed with water, air-dried and photographed. The results of Westerntransfers indicated that the purified material possessed multipleantibody-binding sites.

6.9.4. Enzyme Immunoassay

Immunoaffinity-purified ductal carcinoma antigen was quantitated using asandwich-type of immunoassay procedure in which antigen bound onto asolid-phase antibody was detected using an enzyme-labeled secondarylayer of antibody essentially described in Sec. 6.8.1.

Solid-phase antibody was prepared by incubating McAb F36/22 in plasticmicro-wells (Nunc I Immunoplates (GIBCO, Grand Island, N.Y.)! overnightat 4° C. McAb (10 μg/ml) was dissolved in 50 mM carbonate-bicarbonatebuffer, pH 9.6, prior to the coating procedure. After blockingnon-specific protein binding sites on the plastic using a standard assaybuffer PBS pH 7.0 containing 1% (v/v) nonimmune murine serum and 1%(w/v) bovine serum albumin, or using borate ions!, the plates werewashed twice with assay buffer and used for the assay procedure.

Antigen-containing samples (sera, malignant effusions or chromatographyfractions) were diluted in assay buffer and allowed to incubate withsolid phase antibody for 1.5 hours at 37° C. Following a wash procedure,McAb F36/22 conjugated to horseradish peroxidase (Sigma, Type VI) wasapplied to each well (final concentration 0.5 μg/ml) and incubated for afurther 1.5 hours at 37° C. After several washes, enzyme substrate wasadded to each well for 0.5 hours at room temperature. Substratecontained 0.4 mg o-phenylenediamine/ml of pH 5.0 citrate buffercontaining 0.003% hydrogen peroxide. The enzyme reaction was stoppedupon addition of 50 μg of 2N sulfuric acid, at which time opticalabsorbance (488 nm) was measured using a plate reader (Fisher ScientificCo. Pittsburgh, Pa.).

Specificity of immunoassay was examined by substituting the followingantibody reagents as solid phase for McAb F36/22: McAb M7/105, distincttumor antigen specific Papsidero et al., Cancer Res. 43:1741-1747(1983)!; McAb F5, prostate antigen specific Papsidero, Hybridoma 2:139-147 (1983)!; and nonimmune murine serum. No dose response wasobserved when using these antibodies or when purified carcinoembryonicantigen (8 μg/ml) was used as antigen.

The high molecular weight component identified supra was effective inproducing a dose response curve when using the enzyme immunoassayprocedure as described below and in FIG. 3. Interestingly, borate ionsacted to depress antigen activity, suggesting that carbohydrate may bepresent in or near the antibody-combining sites since borate formscomplexes with cis-glycol groups of carbohydrates. While the PASstaining procedure indicated the presence of periodate-susceptiblecarbohydrates in the antigen, controlled periodate oxidation did notdiminish antigen activity. Since cis-glycols are also the primary pointof attack by periodate, terminal sugars of oligosaccharides may not beinvolved in the antigenic sites. Considering this latter observation,borate most likely interferes with the steric or ionic hindrance of theantibody approach to the epitope.

The format of this assay depends upon the occurrence of multipleantibody-combining sites on ductal carcinoma antigen, thus permitting asandwich-type of procedure. Generally, monoclonal antibodies react withsingly expressed determinants and therefore this procedure cannot beused for detection of any antigen.

6.9.5. Physical Characteristics of Purified of Antigen

Physical characteristics of purified antigen included a single, majorisoionic point near pH 4.2, a symmetrical high molecular weight peakupon molecular sieve chromatography and a broad pattern of migrationupon two-dimensional immunoelectrophoresis.

Isoelectric focusing was done as described by Maidment et al. J. Immun.Meth. 35:297-306 (1980)! using either pH 3-10 or pH 3-5 ampholines.After isoelectric focusing of antigen (5 μg) polyacrylamide gels weresectioned into 3 mm segments and individually placed in stoppered tubescontaining 1 ml of degassed water. After 24 hours at 4° C., the contentsof each vial were monitored for pH and antigen content using the enzymeimmunoassay described supra (absorbance at 488 nm). Broad-range gelsdemonstrated the occurrence of a single major peak of immunoreactivity.Narrow-range gels (pH 3-5) indicated a similar isoionic point at pH 4.2.

Two-dimensional immunoelectrophoresis of immunoaffinity-purified ductalcarcinoma antigen was performed as described by Weeke (1973, In A Manualof Qantitative Immunoelectrophoresis. Methods and Applications (eds.Axelson, N. H., Kroll J. and Weeke B., pp. 47-59, Universitetsforlaget,Oslo). Ten microgram amounts of antigen were subjected toelectrophoresis with second-dimension agarose gels containing McABF36/22 as ascites or nonimmune murine serum at a final concentration of50 μg/ml. Immunoprecipitation reactions were visualized on dried gelsusing Coomassie blue R-250 protein stain. The broad pattern of migrationobserved upon two-dimensional immunoelectrophoresis confirmed theexistence of multiple antibody-combining sites, as suggested fromresults using the sandwich type radioimmunoassay procedure supra.

Purified antigen was also subjected to isopycnic ultracentrifugation.Preparations of purified antigen (1 μg) in PBS were brought to 1.45 gcesium chloride/ml in the presence or absence of 4M guanidinehydrochloride. Samples were then subjected to 1.5×10⁶ g in a BeckmanSW-27 rotor at 10° C. for 72 hours. Fractions of 0.4 ml each werecollected and assessed both for antigen content by enzyme immunoassay(absorbance at 488nm) and density using an analytical balance. Purifiedantigen showed a density of approximately 1.3 g/ml in the presence of 4Mguanidine hydrochloride, and 1.45 g/ml in its absence. Densitymeasurements were consistent with those commonly observed for mucinsassociated with cervix and colonic mucosa Carlstedt et al., Biochem. J.211:13-22 (1983); Hascall and Kimura, Meth. Enzym. 82:769-800 (1982)!.

6.9.6. Lectin Binding Ability of Purified Antigen

Purified antigen (0.4 μg) was allowed to react with excess amounts (200μl ) of 50% (v/v) suspensions! of the following lectins at 4° C. for 18hours: concanvalin A-, lentil lectin-, wheat germ- and peanutagglutinin-Sepharose (Sigma). Subsequently, serial dilutions of thesupernatants were assayed for antigen content. Results were expressed aspercentage bound as compared to control non-derivatized Sepharose.Although rich in carbohydrate, as demonstrated by reactivity with Schiffreagent, purified antigen showed a preferential reactivity with wheatgerm lectin (Table VI). A low or negligible interaction was noted tooccur with concanavalin A, peanut agglutinin and lentil lectin.Therefore, carbohydrate groups available for such interaction were nothighly heterogeneous in composition. Since mucin-like glycoproteinscommonly possess a limited number of oligosaccharide units which arehighly repetitive, these results were not unexpected Hascall and Kimura,Meth. Enzym. 82:769-800, (1982)).

                  TABLE VI    ______________________________________    REACTIVITY OF IMMUNOAFFINITY-PURIFIED DUCTAL    CARCINOMA ANTIGEN WITH LECTINS.sup.a    Solid phase      % bound    ______________________________________    Control.sup.b     0    Concanavalin A    19 ± 14    Wheat germ lectin                     95 ± 6    Lentil lectin    12 ± 4    Peanut agglutinin                      8 ± 7    ______________________________________     .sup.a Four preparations of antigen were tested and results indicate the     mean value ± SEM. McAb F36/22 derivatized to Sepharose served as a     positive control (60 ± 15% bound). No significant binding was observed     with the following antibody adsorbents: McAb M7/105  distinct tumor     antigen, Papsidero et al., Cancer Res.)!, McAb F5  prostate antigen     specific, Papsidero et al., Hybridoma)! and polyclonal rabbit antiserum t     whole human serum.     .sup.b Negative control, nonderivatized Sepharose.

6.9.7. Effects of Physical Treatment, Chemical Modification and Enzymeson the Binding of McAb F36/22 to Purified Antigen

Purified antigen (1 μg) was subjected to heat treatment (60° C. for 5hours) in the presence or absence of 0.1N NaOH or 0.1N HCl. Treatmentsusing periodic acid were performed as follows: immunoaffinity-purifiedantigen was dissolved in periodic acid (50 mM in 0.2M acetate, pH 4.0).The mixture was incubated for 24 hours at 4° C., at which time residualperiodate was removed by addition of 50 μl glycerol 10% (v/v)! for 4hours at 4° C. Each treatment was terminated by addition of cold enzymeassay standard buffer (PBS containing murine serum and albumin) andresidual antigen content was determined as described supra using theenzyme immunoassay (absorbance at 488 nm).

As shown in FIG. 4, the activity of purified antigen was not affectedupon exposure to acid or after heating. In contrast, no antigen activitywas detectable following treatment with base.

The effects of proteolytic enzymes and glycosidase were also determined.Proteolytic enzymes were tested by incubating purified antigen withenzyme (mass ratio of 100:1-antigen:enzyme) in a final volume of 0.1 mlfor 18 hours at 37° C. Reactions were stopped by addition of proteinbuffer prior to enzyme immunoassay to determine residual antigencontent. Control tubes contained identical amounts of antigen in proteinbuffer in which instance the proteolytic enzyme to be studied was addedimmediately before the immunoassay procedure. The following enzymes, andappropriate buffer systems utilized, where tested: pepsin(Boehringer-Mannheim) (PBS); trypsin (Sigma) (PBS); papain (Worthington)(Tris, pH 7.2/10 mM cysteine/1 nM EDTA); and pronase(Boehringer-Mannheim) (PBS).

Treatments using glycosidase enzymes were performed similarly, exceptthat incubation with antigen (final concentration 25 ng/ml) was allowedto proceed for 72 hours at 37° C. The following enzymes were used, alongwith the respective buffer system employed: 0.02 mU/ml neuraminidase(Boehringer-Mannheim) (acetate buffer, pH 5.0, containing 1 mM MgCl₂);740 mU/ml β-galactosidase (Sigma) (PBS containing 1 mM MgCl₂); and 2mU/ml α-mannosidase (Sigma) (PBS) final concentrations!.

Each of these treatments failed to decrease activity of the purifiedantigen. These data suggest that antibody combining sites are either notspecifically hydrolyzed by these reagents or may reside at siteimpervious to enzyme degradation, possibly due to a high carbohydratecontent.

6.10. In Vitro Immunohistological Applications of Monoclonal AntibodyF36/22 6.10.1. Immunoperoxidase Staining of Tumor Specimens by McAbF36/22

In a first set of experiments, sections of formalin-fixed andparaffin-embedded human tumor tissues were used for immunoperoxidasestaining as described previously Heyderman, E. and Neville, A. M., J.Clin. Path., 30:138-140(1976)!. Briefly, hydrated sections were treatedwith 20% pre-immune rabbit serum and then incubated with 100 μl F36/22hybridoma culture fluid (7.5 μg antibody/ml) for 1 hour at roomtemperature. Following 2 washes in D-PBS, peroxidase-conjugated rabbitantibodies against murine immunoglobulin (1/30 dilution; AccurateChemical Corp.) were applied for 1 hour and the slides further washed.Antibody-enzyme conjugates were pre-adsorbed with solid-phase humanimmunoglobulin to avoid spurious binding due to the possible presence ofhuman globulin in tumor specimens. Enzyme activity was revealed using adiamino-benzidine/H₂ O₂ substrate Heyderman and Neville, (1976), supra!.For some experiments, unfixed cryostat sections of fresh breast tumorswere used to assess antibody reactivity.

Overall, 17 out of 22 specimens of intraductal breast carcinoma showedthe presence of antigen recognized by McAb F36/22 (TABLE VII). Severalspecimens of non-mammary tumors also shows immunoperoxidase staining.However, as summarized in TABLE VII, these specimens produced a weakstaining intensity and/or low frequency of positive reactions. Thiscross-reactivity was restricted to adenocarcinomas; other tumor types(sarcoma, lymphoma, myeloma) were consistently negative. Benign lesionsof the breast were further tested with the immunoperoxidase techniqueusing F36/22; these included 3 fibroadenomas and 6 fibrocystic disease.Two specimens each showed positive staining reaction at the apicalmembrane portion of ductal elements. Sections obtained from normalpancreas (n=4), prostate (n=5), colon (n=3), liver (n=4), spleen (n=2)and skin (n=3) were negative, as was a specimen of DMBA-induced mammarycancer of the rat.

                  TABLE VII    ______________________________________    IMMUNOPEROXIDASE STAINING OF TUMOR    SPECIMENS USING MONOCLONAL ANTIBODY F36/22.sup.a               Number of Specimens                         Strongly Weakly    Tumor Type   Tested  Positive.sup.b                                  Positive.sup.c                                         Negative    ______________________________________    Breast Carcinoma                 22      10       7      5    Colon Carcinoma                 10      1        2      7    Lung Carcinoma                 3       0        1      2    Uterus Carcinoma                 10      1        3      6    Liver Carcinoma                 2       0        0      2    Thyroid Carcinoma                 1       0        0      1    Bladder Carcinoma                 6       0        0      6    Prostate Carcinoma                 5       0        1      4    Pancreas Carcinoma                 2       0        0      2    Lymphosarcoma                 3       0        0      3    Lymphoma     2       0        0      2    Plasmacytoma 1       0        0      1    Melanoma     2       0        0      2    Astrocytoma  2       0        0      2    ______________________________________     a. All specimens examined represent formalinfixed and paraffinembedded     tissues examined using monoclonal antibody F36/22 at a concentration of     7.5 μg/ml.     b. Greater than 10% of the tumor cells exhibiting intense cytoplasmic     staining.     c. Less than 10% of the tumor cells exhibiting staining or exhibiting a     weak staining reaction limited to the luminal surface of ductal elements.

In an expanded investigation of the in vivo tissue distribution of theepitope recognized by McAb F36/22, immunoperoxidase staining wasperformed on extra-mammary tumor specimens using a variation of thetechnique described supra.

Paraffin-embedded blocks of tissue were obtained from the PathologyDepartments of St. Joseph's Intercommunity Hospital and Roswell ParkMemorial Institute, Buffalo, N.Y. All tissues were fixed immediately in10% buffered formalin, embedded in paraffin, and sectioned at 3 to 4 μmfor these studies. The sections were collected on slides pretreated withovalbumin and glycerine and heated at 70° C. for 5 minutes to increasethe adherence of the sections to the glass slides.

Hydrated sections were treated with 10% preimmune rabbit serum in 1%ovalbumin and then incubated with 100 μl of hybridoma culture fluid (10μg antibody/ml) for 90 minutes at room temperature. Following washes inD-PBS containing 0.01% Nonidet P-40, peroxidase-conjugated rabbitantibodies against murine immunoglobulin (1/30 dilution; AccurateChemical Corp.) were applied 45 minutes. Human immunoglobulin,convalently-linked to Sepharose beads was used to pre-adsorb theantibody-enzyme conjugate in order to prevent binding to humanimmunoglobulin that might be present in the tumor specimens. In mostcases, enzyme activity was revealed using diamino-benzidine/H₂ O₂substrate in phosphate buffer, pH 6.3. In very sanguinous tissues whichhave large amounts of endogenous peroxidases, amino-ethyl-carbazole (4mg/ml; Sigma)/ H₂ O₂ was used as the substrate to better ascertainspecific binding. In addition to the normal control substitutionexperiments, which included PBS in place of the primary antibody,peroxidase-conjugated antibody alone and culture fluid from a myelomacell line (FLOPC-21), which secreted an irrelevant IgG3 antibody, wereused. The intensity of the immunoreaction-product was scored using a 0to +++ scale for TABLE VIII, however, in order to facilitate statisticalevaluations of the staining intensity data, corresponding numericalvalues of 0 to 3 were utilized.

The results of immunoperoxidase staining on a large group ofextramammary tumors are shown in TABLE VIII. A varying incidence ofantigen expression was observed for these specimens. The majority(54/58) of immuno-positive tumors were histologically classified asadenocarcinomas and no detectable antigen was expressed by othercarcinoma types, including basal cell carcinoma, bronchogenic carcinoma(squamous, large cell and oat cell), squamous cell carcinomas of therectum and endocervix, malignant melanocarcinoma, esophageal carcinoma(squamous and adenocarcinoma) and squamous cell carcinoma of thebladder. The other 4 positive tumors were transitional cell tumors ofthe bladder and renal pelvis. The predominant location of staining inthe majority of these positive tumors was the apical luminal surface ofthe epithelial cells, although the intensity of staining was usuallylower than that observed for breast carcinomas (TABLE VIII).

For several carcinoma sites, including colon, pancreas, jejunum,stomach, prostate and ovary, the adjacent tissue was consistentlynegative for expression of antigenic determinant, but the tumor waspositive. Of additional interest, a few specimens of pancreas and coloncarcinomas showed expression of the antigenic determinant in theepithelial elements of the adenocarcinoma and also in apparently normaltissue directly adjacent to the carcinoma, whereas normal tissue furtherdistal to the tumor was negative. No detectable reactivity with a largepanel of tumors of mesenchymal origin was observed (TABLE VIII),including lymphosarcomas, myelomas, astrocytomas, leukemias andsarcomas.

                  TABLE VIII    ______________________________________    IMMUNOPEROXIDASE STAINING OF    EXTRA-MAMMARY TUMORS WITH McAb F36/22                No.      Pre-                Staining/                         dominant        x % Positive                No.      Location Intensity                                         cells    Tissue Examined                Examined of Stain.sup.a                                  of Stain.sup.b                                         (range).sup.c    ______________________________________    Gastric Carcinoma                 5/10    S        + to ++                                         40 (10-60)    Jejunal Carcinoma                1/2      S        +      30    Colonic Carcinoma                3/9      S        + to ++                                         60 (50-80)    Cholangic Carcinoma                6/7      S        + to ++                                         60 (30-90)    Pancreatic Carcinoma                 4/10    S        +      40 (10-90)    Thyroid Carcinoma                1/2      S        +      40    Endocervical                1/2      S        + to ++                                         80    Carcinoma    Endometrial  9/13    S/C      + to +++                                         70 (30-90)    Carcinoma    Fallopian Tube    Carcinoma   0/2    Ovarian Carcinoma                15/15    S        + to +++                                         80 (70-90)    Prostatic Carcinoma                2/8      S        +      10 (20-20)    Renal Carcinoma                3/6      S        + to +++                                         40 (20-60)    Bronchogenic                 5/13    S/C      + to +++                                         70 (50-90)    Carcinoma    Ovarian Cystadenoma                3/3      S/C      + to +++                                         40 (20-80)    Bladder Carcinoma                2/5      C        ++     30 (20-40)    ______________________________________     .sup.a S = Marginal and/or luminal surface; C = cytoplasmic; S/C = surfac     and/or cytoplasmic.     .sup.b Intensity scale: - negligible; + weak; ++ moderate; +++ strong.     .sup.c Percent of positive staining cells was estimated by observing the     number of positive cells in replicate 100 × fields.     .sup.d No staining was observed in the following extramammary tumor     specimens (the number in parenthesis indicates the number of samples     examined): esophageal carcinoma (5); hepatic carcinoma (2); parotid gland     carcinoma (3); submaxillary gland. carcinoma (1); seminoma (2); malignant     melanoma (3); basal cell carcinoma (4); squamous cell carcinoma (3);     sebaceous gland carcinoma (1); leukemias (4); multiple myeloma (3);     meningioma (2); astrocytoma (3); lymphosarcoma (6); Kaposi's sarcoma (1);     fibrosarcoma (3); glomus tumor (1); carcinoid (2); teratoma (4);     hidroadenoma (4); myoxma (3); neuroma (3); hemangioma (2); hamartoma (2);     lymphangioma (1); lipoma (4); trichoepithelioma (3); fibroxanthoma (2);     nevus (benign) (1);

6.10.2. Immunoreactivity of McAB F36/22 with Normal Mammary Tissue,Membrane Preparations and Milk

McAb F36/22 was tested for its ability to react with mammary membranesusing adsorption analysis and direct binding radioimmunassay procedures.To obtain tissue membranes, normal breast tissue was homogenized inD-PBS containing protease inhibitors (0.5 mM PMSF and 1.0 mMepsilon-aminocaproic acid). All steps were performed at 4° C. Celldebris and nuclei were pelleted at 2,000 ×g and the supernatant wasfurther centrifuged at 40,000 ×g to obtain a crude membrane fraction.The membranes were washed three times with D-PBS and adjusted to 2 mgprotein/ml.

Membranes from human milk were isolated as follows: the cream fractionwas washed 5 times with D-PBS to remove whey proteins and was quicklyfrozen. The cream was then slowly thawed, thereby increasing the ruptureof membrane globules, and then was shaken vigorously in D-PBS containing1 mM MgCl₂ When butter formation was observed, the suspension wascentrifuged at 100,000 ×g for 1 hour. Membranes were resuspended inD-PBS at 2 mg protein/ml.

For direct binding measurements, 50 μl samples of membrane preparationswere added to wells of glutaraldehyde-sensitized microtiter plates.After adhering overnight, the wells were washed twice with 0.25% glycineand then 4 times with D-PBS containing 1% bovine serum albumin. Theassay was performed in triplicate using 100 μl samples of McAb F36/22which were incubated with the membranes for 90 minutes at roomtemperature. The remainder of the assay was performed as in Section6.5., for CS-RIA procedure.

Membrane fractions obtained by the foregoing procedures from bothtissues and human milk fat globules were examined along with the frozensections of normal mammary tissues. As shown in TABLE IX, antibodyF36/22 directly bound to all membrane preparations examined, and wasable to be adsorbed by these specimens. Immunoperoxidase data confirmedthe identity of the reactive antigen as a component of the ductalepithelial surface membrane.

                  TABLE IX    ______________________________________    IMMUNOREACTIVITY OF NORMAL MAMMARY    TISSUES AND MEMBRANE PREPARATIONS                   Reactivity with Antibody    Specimen       F36/22    ______________________________________    Mammary Tissues.sup.a                   +    Membranes.sup.b    Tissues        +    Milk Fat Globule                   +    ______________________________________     a. Cryostat sections of frozen mammary tissues (n = 3) were examined by     standard immunoperoxidase staining techniques (see Section 6.10.1).     b. Membrane preparation at 2 mg protein/ml were tested for their     immunoreactivity using absorption analysis and directbinding     radioimmunoassay (see Section 6., and 6.5., respectively).

A second set of experiments testing the reactivity of McAb F36/22 withnormal tissues was performed using the immunoperoxidase stainingtechnique. The results are summarized in TABLE X.

In general, positive staining was restricted to the epithelial elementsof a few exocrine glands and their associated ducts. These includedsweat glands, sebaceous glands, and endometrial glands. Other tissueswith positive staining were respiratory alveoli, ductuli efferentes ofepididymis, fallopian tube and the distal tubules and collecting ductsof the kidney. These tissues exhibited a very weak, apical luminalstaining.

There was no relationship between the degree of positive staining inendometrial glands and the different phases of the menstrual cycle.Additionally, the tissue components of ovary, colon, stomach, pancreas,prostate, and gall bladder did not contain detectable levels of theantigen determinant recognized by McAb F36/22, whereas theadenocarcinomas of these histotypes did express detectable levels to avarying incidence. The bile ducts of liver, the acinar cells of thepancreas, and the serous demilunes of the salivary gland were allnegative for the expression of the epitope.

TABLE X Immunoperoxidase Staining of Normal Tissue with McAb F36/22^(a)

Positive Staining Restricted to Epithelial Elements of

The globules and cell surfaces of sebaceous glands (5/5)^(b), alveolarepithelium of lung (4/5), sweat glands and associated ducts (5/5),ductuli efferentes of epididymis (3/3), endometrial glands and ducts(8/8), fallopian tube (4/5), distal tubules and collecting ducts ofkidney (4/4).

No Staining of Tissue Elements of

Esophagus (2)^(c), stomach (3), small intestine (3), appendix (3), colon(5), pancreas (4), gall bladder (2), liver (5), parotid gland (2)submaxillary gland (3), thyroid (3), adrenals (2), spleen (3), tonsil(2), lymph node (2) bone marrow (3), prostate (5), ovary (15), testes(4), nervous tissue (5), skin (5), myocardium (3), trachea (1) andbladder (2).

a. Normal tissues distal to tumors examined in the present study werealso evaluated for expression of this antigen determinant, but thesedata were not included in the above numbers.

b. No. of tissues staining/No. of tissues examined.

c. The No. in parentheses represents total No. examined.

6.10.3 Immunoperoxidase Staining of Human Breast Tissues and Tumors

Non-malignant and primary breast tumor specimens were obtained atsurgery. Freshly obtained autopsy specimens of normal tissues anddistant metastatic lesions were also examined. All tissues were fixedimmediately in 10% buffered formalin, embedded in paraffin, andsectioned at 3 to 4 μm for these studies. The sections were collected onslides pretreated with ovalbumin and glycerine and heated at 70° C. forthese studies. The sections were collected on slided pretreated withovabumin and glycerine and heated at 70° C. for 5 minutes to increaseadherence of the sections to the glass slides. The immunoperoxidasestaining technique described for the expanded investigation in Section6.10.1. was employed.

The results of tissue staining for normal resting breast, benign andmalignant breast tumors are presented in TABLE XI. In general, thenormal resting breast parenchyma exhibited delicate, but weak,immunoperoxidase staining restricted to the apical luminal surface ofepithelial cells. Benign tumors of the breast displayed similar locationof the staining reaction, except that the intensity of the reaction wasgenerally greater. All gynecomastia specimens examined showed positiveimmuno-reactivity consistent with that observed in the benignconditions. Primary breast tumors, however, displayed significantvariations in the percentage of cells stained in any given tumor, in theintensity of the staining reaction and, and in the location of theimmuno-reaction. As is summarized in TABLE XI, expression or lack ofexpression of the antigenic determinant did not correlate with the gradeof the tumor. However, McAb F36/22 predominantly stained the surfacemembranes of luminal epithelial cells in well-differentiated carcinomasand those cancer cells bordering lumina within the in situ and comedotumors. Poorly-differentiated tumors, when stainable, most oftenexhibited focal cytoplasmic staining. Intensity of the reaction productvaried from specimen to specimen (TABLE XI).

                  TABLE VIII    ______________________________________    INDIRECT IMMUNOPEROXIDASE STAINING OF    HUMAN BREAST TISSUE WITH McAb F36/22                No.      Pre-                Staining/                         dominant        x % Positive                No.      Location Intensity                                         cells    Tissue Examined                Examined of Stain.sup.a                                  of Stain.sup.b                                         (range).sup.c    ______________________________________    Normal    Resting Breast                4/4      S        +      70 (40-90)    Non    Malignant Lesions    Fibrocystic disease                6/6      S        + to +++                                         70 (40-90)    Fibroadenoma                6/6      S        + to +++                                         80 (70-90)    Cystosarcoma    Phylloides  1/1      S        +++    90    Papilloaa   2/2      S        + to ++                                         90 (80-100)    Gynecomastia                5/5      S        + to +++                                         70 (20-90)    Malignant Lesions    Ductal Carcinoma:    Tubular type                0/2    Moderately diff.                4/4      S/C      + to +++                                         30 (20/50)    Poorly diff.                10/13             + to +++                                         70 (30-90)    Comedo type 5/5      S/C      + to +++                                         80 (50-90)    Infiltrating    Ductal Carcinoma:    Well diff.  3/4      S        + to +++                                         50 (40-80)    Well-Mod. diff.                1/1      S/C      +++    90    Moderately diff.                 7/10    S/C      + to +++                                         60 (10-90)    Mod.-Poorly diff.                8/9      S/C      +++    90 (80-100)    Poorly diff.                17/18    C        + to +++                                         60 (40-90)    Mucoid Carcinoma                3/3      S/C      ++ to +++                                         90 (80-100)    Lobular Carcinoma                8/9      C        + to +++                                         80 (50-90)    Medullary Carcinoma                1/1      C        ++ to +++                                         60    Squamous Cell    Carcinoma   1/1      C        + to ++                                         50    Lymphouarcoma                0/1    Metastatic    Lymph nodes  5/12    S/C      + to +++                                         50 (30-90)    ______________________________________     .sup.a S = marginal and/or luminal surface; C = cytoplaasmic; S/C =     surface and/or cytoplasmic.     .sup.b Intensity seale: - negative; + weak; ++ moderate; +++ strong.     .sup.c Percent of positive staining wells was estimated by observing     replicate 100 × fields of view.

6.10.4. Effect of Varying McAb F36/22 Concentration and incubation Timeson Immunoperoxidase Staining Results

To determine if the lack of detectable antigen expression in some tumorswas merely reflecting an inadequate antibody concentration, thefollowing study was performed. Two reference breast carcinomas whichwere known to be positive for the expression of the epitope recognizedby McAb F36/22 were serially sectioned. Serial dilutions of McAb F36/22(initial concentration: 10 μg/ml) were incubated with the sections whichthen were evaluated by immunoperoxidase staining. The highest dilutionwhich exhibited immunopositivity when compared to the initialconcentration was determined.

Increasing the concentration of McAb F36/22 from 10 to 20 μg/ml did notsignificantly increase the percentage of tumors exhibiting positiveimmunoperoxidase staining. Furthermore, antibody at 200 ng/ml wascapable of staining greater than 95% of the original immunopositivecells. Thus, the present experiments were performed at antibody excess.Increasing the incubation times also did not affect the sensitivity ofthe assay.

6.10.5. Estrogen Receptor Levels and Immunoperoxidase Straining

To determine if the staining patterns recognized by McAb F36/22 wererelated to estrogen receptor status, 60 breast tumors of known estrogenreceptor levels were stained by immunoperoxidase techniques aspreviously outlined. The estrogen receptor levels of these specimenswere determined at the Buffalo General Hospital, Department of ClinicalChemistry, as described by Rosen, P.P. et al. Cancer Res.35:3187-3194(1975)!.

Breast cancer specimens of known estrogen receptor level (n=60) 42ER-rich, 18 ER-poor, were examined by immunoperoxidase techniques. Thepercentage of positive cells, the mean ±S.D., (69±30) in the ER-richcarcinomas was greater (p less than 0.001) than that exhibited inER-poor cancers (33±37). In addition, the intensity of the stainingreaction associated with ER-rich cancers (2.1±0.8) was significantlygreater (p less than 0.001) than the reaction product observed inER-poor carcinomas (1.0±0.9), as assessed using the semi-quantitativetechnique of immunoperoxidase staining.

6.11. In Vivo Applications of Monoclonal Antibody F36/22 6.11.1.Experimental Induction of Solid Tumors in Mice

Human tumor cells were mechanically-harvested from tissue culture flasksand washed with serum-free RPMI-1640 medium.

Cell preparations showing greater than 90% viability, as assessed withtrypan blue, were used to produce solid tumors in mice. Athymic femaleSwiss nude (nu/nu) mice were given subcutaneous injections of 0.2 mlcontaining 4-10×10⁶ viable tumor cells. MCF-7 and BT-20 (human breastcarcinoma), Chago (human lung carcinoma) and Daudi (lymphoblastoidcells) were used. Tumor dimensions were subsequently monitored at dailyintervals using calipers and the tumor volume was calculated using aformula described by Kovnat, A. et al. Cancer Res., 42:3969-73(1982)!.

6.11.2. McAb F36/22 Targeting: in Vivo Tumor Localization

To evaluate whether immunotherapeutic effects could be attributed to adirect interaction between tumor and antibody, targeting experimentswere performed to determine the tissue distribution of injectedradio-antibody.

For one set of antibody targeting experiments, purified immunoglobulinswere labelled with iodine-125 to a specific activity of 5 ci/mM! Fraker,P. J. and Speck, Jr., J. C., Biochem. Biophys. Res. Commun.,80:849-57(1978)!. Athymic mice bearing human tumor xenografts induced byMCF-7 or Chago cells, were given a single intraperitoneal injection ofradiolabelled immunoglobulin (10-20 μci) on day 0. On day 7, the animalswere exsanguinated by cardiac puncture and tumors and organs wereremoved. After weighing each tissue, radioactivity was measured in awell-type gamma counter. The results were expressed as a localizationratio, i.e., cpm per gram of tissue (tumor or organ) divided by cpm pergram of blood.

As shown in TABLE XII, McAb F36/22 localized to breast tumors in greateramounts per gram of tissue than other organs examined. Effectivetargeting of radio-antibody to breast tumor xenografts indicates adirect interaction between tumor cells and F36/22 monoclonal antibody.This targeting effect was observed with the use of controlimmunoglobulin preparations or against lung carcinoma xenografts (TABLEXII).

                  TABLE XII    ______________________________________    TARGETING OF RADIOLABELLED MONOCLONAL    ANTIBODY (F36/22) IN ATHYMIC    MICE BEARING HUMAN TUMOR XENOGRAFTS.sup.a    Tissue.sup.b     Localization Ratio.sup.c    ______________________________________    Blood            1    Breast Carcinoma (MCF-7)                     3.4 ± 0.5    Lung Carcinoma (Chago)                     0.7 ± 0.1    Lung             1.2 ± 0.3    Liver            0.5 ± 0.2    Spleen           0.4 ± 0.1    Kidney           0.5 ± 0.2    Brain            0.5 ± 0.1    ______________________________________     .sup.a Athymic mice (4 per group) were each implanted with 2 human tumor     xenografts on contralacteral sites of the back. These consisted of breach     (MCF7) and lung (Chago) carcinomas. Xenografts were of similar size at     onset of the experiments (between 5 and 15 mm.sup.3). Each animal receive     between 10 to 15 μCi of radiolabelled monoclonal antibody (F36/22) by     the intraperitoneal route.     .sup.b Tissues and blood were harvested 7 days post injection.     .sup.c Localization Ratio: cpm per gram tissue (tumor or organ) divided b     cpm per gram of blood, (mean ± S.D.).

In a second set of targeting experiments, BT-20 (human breastcarcinoma), and Chago (human lung carcinoma) were used. The growthpattern of the xenografts was carefully monitored in order toconsistently work with similar sizes of growing tumors. In vivo tumorlocalization was performed essentially the same as above. Twentymicrograms of ¹²⁵ I-labelled McAb F36/22 (specific activity, 1 μCi/μg)was injected intraperitoneally using four groups of nude mice (n=3)bearing either BT-20 or Chago tumors. One group of mice was sacrificedat sequential days after administration of radiolabelled McAb F36/22.Twelve tissues were removed (blood, liver, lung, spleen, heart, skeletalmuscle, skin, kidney, intestine, brain, and tumor), and the tissuedistribution of the labelled antibody was determined with a Gammacounter. Tissue levels were calculated in disintegrations per minute pergram of tissue. Blood levels were taken as the reference background sothat relative antibody uptake into tissues (localization ratio) wasdetermined to be the ratio of disintegrations per minute per gram oftissue divided by the disintegrations per minute per gram of blood. Theuptake of McAb in the BT-20 tumors was 5.7 (±0.4), (mean±standarddeviation) fold greater than the blood background (p less than 0.02), at7 days post injection; whereas no other tissue including Chago tumorsshowed statistically significant activity above blood levels.

In contrast, normal mouse ¹²⁵ I-labelled antibody (γ3 subclass) showedno specific uptake into any tissues or tumors sampled. Significantspecific uptake of McAb F36/22 was also seen in MCF-7 breast carcinoma,which gave a localization ratio of 3.4 (±0.5), as compared to bloodlevels (p less than 0.05).

6.11.3. In Vivo Passive Immunotherapy with McAb F36/22

For one set of therapy studies, mice with progressively growing humantumors (MCF-7, Chago or Daudi) were given a single intraperitonealinjection at day 0 of either monoclonal antibody F36/22 or controlimmunoglobulin. One hundred micrograms were administered in a totalvolume of 0.2 ml sterile PBS. Mean tumor volumes at day 0 were: MCF-7,57mm³ ; Daudi, 59 mm³ ; Chago, 105 mm³. Post-therapy tumor volumes weretaken at daily intervals and statistical evaluations were performedusing Student's t-test. Based upon these measurements the tumors werescored as either progressive, static, or regressive. For each experimentmice were monitored for one week, at which time tumor specimens wereexcised for histological examination. Results obtained using calipermeasurements indicated that breast carcinoma xenografts treated withantibody F36/22 had regressed to approximately 15% of theirpre-treatment volumes (57 to 7mm³) at day 7. As expected with the use ofa murine host, non-immune mouse IgG produced no such effect at the samedose level. The therapeutic effect of antibody F36/22 was evident by day4 post-therapy, when a significant (p less than 0.01) decrease(approximately 75%) in breast tumor volumes was observed. Replicategroups of antibody-treated mice which were observed for 2 weeksdemonstrated breast tumor xenografts exhibiting no detectable capacityfor regrowth during this period. Histological examination of breasttumors obtained 7 days post-therapy indicated a significant amount oftumor cell necrosis accompanied by initial stages of fibrosis. Incontrast, the histological picture of control tumors treated withnon-immune IgG was consistent with the normal histology of MCF-7 breastcarcinoma xenografts.

The therapeutic specifity of antibody F36/22 was evaluated against twonon-mammary human tumor xenografts implanted in nude mice. For theseexperiments a slow growing lymphoma (Daudi) and rapidly multiplying lungcarcinoma (Chago) were used. Results indicate that antibody treatmentwas ineffective in causing regression of these tumors within 4 dayspost-therapy. Further, at 7 days post-therapy, the lymphomas were atover 300% of their day 0 volume (from 59 to 222 mm³ at days 0 and 7,respectively). A similar progression of lung carcinoma xenografts wasalso observed (105 and 706 mm³ at days 0 and 7, respectively).

The effects of McAb against tumors in vivo were tested in anotherexperiment. One group of mice (n=4), bearing human breast carcinoma(BT-20) were inoculated intraperitoneally with 100 μg of purified Ey, P.L. et al. Immunochem., 15:429-436 (1978)! McAb F36/22. A significantreduction (40%) of tumor volumes was seen for each of the individualmice one day following injection of McAb F36/22 (p less than 0.001 foreach tumor as compared to pre-treatment volumes) Tumor volumes weremeasured daily with precision caliper and calculated by the 3 diameterproduct. No tumor volume reduction occurred during the 7 days followingadministration of the control antibody.

In order to test reproducibility and statistical significance of theMcAb therapy, further experiments were performed. After 3 weeks to 2months post-implantation, 26 BT-20 breast tumors of sizes ranging from12 to 199 mm³ (mean, 61 mm³) were produced. These tumor-bearing micewere injected with either 100 μg of purified McAb F36/22 or one of 2identically-purified control antibodies (one a γ3 myeloma protein withno known'specificity, the other a γ2a complement-fixing McAb with nocell binding specificity to BT-20 cells). Fourteen mice with tumorsranging from 18 to 199 mm³ (mean±SD, 62±46 mm³) were each injectedintraperitoneally with 100 μg of McAb F36/22 in PBS, and 12 mice withtumors ranging from 12 to 149 mm³ (mean±SD, 61±42 mm³) with the sameamount of control antibody (7 of the control mice with the γ3 proteinand 5 of the control mice with the γ2a McAb). Therapy of breast cancerwith McAb F36/22 resulted in a rapid reduction in tumor size. Tumorregression continued for about 3-4 days post-therapy and thereafter thetumors remained at approximately 25 percent of their pretreatmentvolume. In comparison, breast tumors were at 115 (±37) percent oforiginal volume after treatment with control immunoglobulin.Measurements of the control antibody-treated group showed 6 mice hadslowly enlarging tumors; 2 tumors were stable and 4 tumors were slowlyregressing in volume over the 7 days of the experiment. Similarvariations of growth patterns were typically seen in breast carcinomaxenografts as thoroughly described by others Ozzelo, L. and Sordat, M.,Eur. J. Cancer, 164:553-559 (1980)!. The McAb F36/22 produced noregression of Daudi lymphoid tumors or fast growing Chago lungcarcinomas. The BT-20 tumors were significantly reduced in volume (pless than 0.001) as compared to controls at 24 hours post-therapy andremained at a decreased volume for the course of the 7 day experiment.The mice with closely matched tumor sizes were segregated into groupsconsisting of small and large BT-20 tumors, in order to judge thepossible influence of pre-treatment tumor size on the amount of tumorvolume reduction following therapy. The first group consisted of 11 micewith tumors ranging from 26-49 mm³. The second group had 9 mice withtumor volumes ranging from 60-88 mm³. The smaller tumors decreased to5(±3) mm³, 34 days after McAb F36/22 treatment as compared to 51(±25) mmfor control antibody treated mice (p less than 0.001) (TABLE XIII). Thelarger tumors were reduced to 25(±18) mm³ 4 days after McAb F36/22therapy, as compared to 74(±20) mm³ for controls (p less than 0.005)(TABLE XIII). No statistically significant difference was observed inthe percentage of tumor volume reduction between the larger and smallertumor groups.

Tumor histology which was taken before therapy and at 1, 3 and 5 dayintervals after the injection with 100 μg of McAb 36/22, demonstratedextensive necrosis of tumor cells on days 1, 3 and 5. On day 1, tumorcell necrosis was most prominent in the central region of the tumor,leaving a semi-circular ring of intact tumor cells (1 to 20 cells thick)at the periphery of the tumor. Acute inflammatory cell infiltration wasalso seen. By day 3, tumor contraction was observed, accompanied by bothchronic and acute inflammatory cells. On day 5 scar tissue activity wasevident along with a predominantly chronic inflammatory pattern of cellinfiltration. These histological changes appear to be very similar tothose observed by others using a polyclonal sera on a virally infectedchemically induced murine sarcoma Ward, E. W. et al., J. Natl. CancerInst. 69:509-515 (1982)!. Extensive tumor cell necrosis was also seenfor MCF-7 breast tumors treated with McAb F36/22 and no histologicalchanges were seen in identically treated Chago tumors as compared tocontrol tumors.

It is apparent that many modifications and variations of this inventionas hereinabove set forth may be made without departing from the spiritand scope thereof. The specific embodiments described are given by wayof example only and the invention is limited only by the terms of theappended claims.

A cell line, F36/22, as described herein was deposited with the AmericanType Culture Collection, Rockville, Md. Mar. 1, 1993 and has beenassigned accession number ATCC No. HB8215. The invention described andclaimed herein is not to be limited in scope by the cell line deposited,since the deposited embodiment is intended as a single illustration ofone aspect of the invention and any equivalent cell lines which producea functionally equivalent monoclonal antibody are within the scope ofthis invention. Indeed, various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are also intended to fall within the scope of the appendedclaims.

                                      TABLE XIII    __________________________________________________________________________    COMPARISON OF PRE-TREATMENT TUMOR SIZE TO McAb F36/22 MEDIATED VOLUME    REDUCTIONS.sup.a           Number of                 Day 0 Range of                           Tumor Volume.sup.b    Antibody           Mice/Group                 Tumor Volume (mm)                           Day 0                               Day 1                                   Day 2                                       Day 3                                           Day 4                                               Day 5                                                   Day 6                                                       Day 7    __________________________________________________________________________    Group I    (small tumors)    F36/22 6     26-42     34 ± 7                               11 ± 8                                   9 ± 6                                       9 ± 5                                           5 ± 3                                               7 ± 3                                                   6 ± 1                                                       9 ± 4    Control (γ3)           5     25-49     38 ± 8                               39 ± 2                                   46 ± 14                                       44 ± 16                                           51 ± 25                                               48 ± 30                                                   45 ± 26                                                       41 ± 20    Level of           --    --        N.S..sup.d                               .001                                   .001                                       .001                                           .001                                               .001                                                   .001                                                       .001    significance    (less than)    Group 2    (large tumors)    F36/22 6     60-80     74 ± 11                               33 ± 16                                   27 ± 15                                       24 ± 20                                           25 ± 18                                               22 ± 16                                                   22 ± 18                                                       20 ± 17    Control (γ3)           3     80-88     84 ± 4                               84 ± 6                                   85 ± 8                                       81 ± 7                                           74 ± 20                                               72 ± 15                                                   69 ± 10                                                       70 ± 14    Level of           --    --        N.S..sup.d                               .001                                   .001                                       .001                                           .001                                               .001                                                   .001                                                       .001    significance    (less than)    __________________________________________________________________________     .sup.a Mice with BT20 tumors were pooled seperately into groups of small     and large tumors, the smaller tumors ranging from 26-49 mm.sup.3, the     larger tumors 60-88 mm.sup.3. Each group was treated with 100 μg of     McAB F36/22 for the experimental mice or 100 μg of control     immunoglobulin for the control mice.     .sup.b All tumor volumes given as x ± SD, in mm.sup.3.     .sup.c p values calculated by Student's t distribution.     .sup.d N.S., no statistically significant difference.

We claim:
 1. A method for detecting the presence of adenocarcinoma,which comprises: screening, wherein screening comprises an immunoassay,a serum, lymph or thoracentesis sample from a patient suspected ofhaving adenocarcinoma to detect the presence of an antigen, wherein theantigen contain an epitope recognized by monoclonal antibody F36/22having ATCC accession number HB 8215 which is a mucin-like glycoproteincharacterized by a molecular weight greater than about 669,000 daltonsas measured by gel filtration chromatography, an isoionic point of aboutpH 4.2 as determined by isoelectric focusing, a density of about 1.45g/ml as determined by isopycnic ultracentrifugation in the absence ofguanidine hydrochloride, in which the presence of the antigen correlateswith the presence of adenocarcinoma.
 2. The method of claim 1, in whichthe immunoassay is an enzyme linked immunoassay.
 3. The method of claim1 which comprises screening a thoracentesis sample.
 4. The method ofclaim 1 which comprises screening a serum sample.
 5. The method of claim1 which comprises screening a lymph sample.